WO2020139857A1 - Nitric oxide-releasing antibacterial polymers and scaffolds fabricated therefrom and methods pertaining thereto - Google Patents

Nitric oxide-releasing antibacterial polymers and scaffolds fabricated therefrom and methods pertaining thereto Download PDF

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WO2020139857A1
WO2020139857A1 PCT/US2019/068412 US2019068412W WO2020139857A1 WO 2020139857 A1 WO2020139857 A1 WO 2020139857A1 US 2019068412 W US2019068412 W US 2019068412W WO 2020139857 A1 WO2020139857 A1 WO 2020139857A1
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compound
group
alkyl
several embodiments
polymer
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PCT/US2019/068412
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English (en)
French (fr)
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Mark H. Schoenfisch
Mona Jasmine R. AHONEN
Lei Yang
Haibao JIN
Evan Scott FEURA
Sara Elizabeth MALONEY
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The University Of North Carolina At Chapel Hill
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Priority to CN201980089631.6A priority Critical patent/CN113383019B/zh
Priority to EP19904463.7A priority patent/EP3902841B1/en
Priority to AU2019414421A priority patent/AU2019414421B2/en
Priority to CA3124673A priority patent/CA3124673A1/en
Priority to JP2021537725A priority patent/JP7645546B2/ja
Publication of WO2020139857A1 publication Critical patent/WO2020139857A1/en

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    • C08B15/00—Preparation of other cellulose derivatives or modified cellulose, e.g. complexes
    • C08B15/02—Oxycellulose; Hydrocellulose; Cellulosehydrate, e.g. microcrystalline cellulose
    • C08B15/04—Carboxycellulose, e.g. prepared by oxidation with nitrogen dioxide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/728—Hyaluronic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/717—Celluloses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/40—Cyclodextrins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/28—Materials for coating prostheses
    • A61L27/34—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
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    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
    • A61L29/04—Macromolecular materials
    • A61L29/043—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
    • A61L29/14—Materials characterised by their function or physical properties, e.g. lubricating compositions
    • A61L29/16—Biologically active materials, e.g. therapeutic substances
    • C—CHEMISTRY; METALLURGY
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    • C08B11/00—Preparation of cellulose ethers
    • C08B11/02—Alkyl or cycloalkyl ethers
    • C08B11/04—Alkyl or cycloalkyl ethers with substituted hydrocarbon radicals
    • C08B11/10—Alkyl or cycloalkyl ethers with substituted hydrocarbon radicals substituted with acid radicals
    • C08B11/12—Alkyl or cycloalkyl ethers with substituted hydrocarbon radicals substituted with acid radicals substituted with carboxylic radicals, e.g. carboxymethylcellulose [CMC]
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
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    • C08B15/00—Preparation of other cellulose derivatives or modified cellulose, e.g. complexes
    • C08B15/05—Derivatives containing elements other than carbon, hydrogen, oxygen, halogens or sulfur
    • C08B15/06—Derivatives containing elements other than carbon, hydrogen, oxygen, halogens or sulfur containing nitrogen, e.g. carbamates
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/10—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing inorganic materials
    • A61L2300/114—Nitric oxide, i.e. NO
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/404—Biocides, antimicrobial agents, antiseptic agents

Definitions

  • the presently disclosed subject matter relates generally to nitric oxide- releasing polymers and scaffolds made therefrom that store and/or release nitric oxide in a controlled manner. Additionally disclosed are methods of synthesis of the same and methods of use of the same as antibacterial agents in methods of treatment.
  • Biofilms are cooperative communities of bacteria encapsulated by an exopolysaccharide (EPS) matrix protecting the bacteria from host immune response and antibiotics.
  • EPS exopolysaccharide
  • Nitric oxide plays a variety of physiological roles as a signaling molecule and, as disclosed herein, can also play significant roles in treating or ameliorating pathophysiology, for example as a therapeutic agent.
  • NO as a therapeutic has heretofore been underused, based at least in part on limited NO payloads of therapeutic compositions, NO release rates that are more rapid than desired, and the lack of targeted NO delivery.
  • NO-releasing polymers and scaffolds methods of producing such polymers and scaffolds, and methods of treating various pathophysiologies using such polymers and scaffolds that leverage enhanced NO-release characteristics and beneficial physical properties, harnessing the abundant potential of NO-releasing pharmacological compounds and compositions.
  • provided herein are compounds and compositions that are highly efficacious as antimicrobials.
  • the polymers and/or scaffolds disclosed herein have advantageous activity as viscosity enhancing agents.
  • the macromolecular structures are polymers. While, in several embodiments, the polymers can be used as dilute solutions (e.g., for vaporization and inhalation, etc.), in other embodiments, the polymers can be self-assembled in solution and/or can be crosslinked to provide scaffolds with advantageous physical properties (including three-dimensional shape, firmness, adhesiveness, and viscosity). In several embodiments, the polymers retain beneficial antimicrobial activity even as gels and viscous liquids.
  • a polymer having structural units along a chain of the polymer is provided.
  • one or more of the structural units is functionalized with one or more instances of each of R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 .
  • R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 can comprise functional units that imbue the polymer with desired properties.
  • R 1 , R 2 , R 3 , R 4 , R 5 , and R6 can comprise NO binding moieties.
  • each instance of R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 along the polymer chain is independently selected from one or more of -OH, -NH 2 , -OCH 3 , -C(O)OH, -CH 2 OH, -CH 2 OCH 3 , -CH 2 OCH 2 CH 2 OH, -OCH 2 C(O)O H, -CH 2 OCH 2 C(O)OH, -CH 2 C(O)OH, -NHC(O)-CH 3 , -C(O)O((CH 2 ) a O) b -H, -C(O)O((CH 2 ) a O) b -(CH 2 ) c H, -C(O)O(C 1-5 alkyl), -C(O)-NH-((CH 2 ) d NH) e -H, -C(O)
  • each instance of a, b, c, d, e, f, g, h, i, j, k, and l is independently selected from an integer of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • each of a, b, c, d, e, f, g, h, i, j, k, and l can be independently greater than 10.
  • each instance of X 1 , X 2 , and X 3 is independently selected from -O-, -S-, -NH-, and C(O)NH-.
  • At least one instance of X 1 , X 2 , and X 3 is a NO donating moiety. In several embodiments, at least one instance of X 1 , X 2 , and X 3 is represented by one of the following:
  • each instance of R 1 , R 2 , R 3 , R 4 , R 5 , or R 6 is independently selected from the group consisting of:
  • each secondary amine of the above structures may be functionalized with a NO donating group as disclosed herein.
  • the polymer (or scaffold made therefrom) has a viscosity of equal to or at least about 10 mPa ⁇ s at 20 °C at a concentration of 5% w/w. In several embodiments, the polymer (or scaffold made therefrom) has a gel firmness of equal to or at least about 1.0 Nm at a concentration of 5% w/w.
  • the viscosity can be greater, such as for example, about 10 mPa ⁇ s, about 20 mPa ⁇ s, about 30 mPa ⁇ s, about 50 mPa ⁇ s, about 60 mPa ⁇ s, about 70 mPa ⁇ s, about 80 mPa ⁇ s, about 90 mPa ⁇ s, about 100 mPa ⁇ s, or any viscosity therebetween.
  • the gel firmness can be greater, for example about 5 Nm, about 10 Nm, about 20 Nm, about 30 Nm, about 40 Nm, about 50 Nm, about 75 Nm, about 100 Nm, or any gel firmness therebetween.
  • the polymer is a biopolymer. In several embodiments, the polymer is a polysaccharide. In several embodiments, the one or more structural units are represented by saccharide unit of Formula I:
  • the saccharide unit is representative of a carboxymethylcellulose structural unit.
  • the structure of Formula I represents a saccharide unit of a hyaluronic acid polymer.
  • the structure of Formula I represents a saccharide unit of a hydroxyethyl cellulose polymer.
  • the structure of Formula I represents a saccharide unit of a methyl cellulose polymer.
  • the structure of Formula I represents a saccharide unit of an alginate polymer.
  • the structure of Formula I represents a saccharide unit of a cyclodextrin ring structure.
  • R 1 , R 2 , and R 3 are independently selected from -OH, -CH 2 OH, -OCH 2 C(O)OH, -CH 2 OCH 2 C(O)OH, -C(O)-O-((CH 2 ) a O) b -H, -C(O)- O-((CH 2 ) a O) b -(CH 2 ) c H, -C(O)-O-(C 1-5 alkyl), -C(O)-NH-((CH 2 ) d NH) e -H, -C(O)-NH-((CH 2 ) d NH) e -(CH 2 ) f H, -C(O)-X 1 -((CH 2 ) g X 2 ) h -(CH 2 ) 1 H, -C(O)-X 1 -((CH 2 ) g X 2 ) h (CH 2 ) 1 H,
  • each instance of a, b, c, d, e, f, g, h, i, j, k, and l is independently selected from an integer of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • each instance of X 1 , X 2 , and X 3 is independently selected from -O-, -S-, -NH-, C(O)NH-.
  • at least one of X 1 , X 2 , and X 3 is represented by one of the following:
  • the carboxymethylcellulose-derived polymer compound has a viscosity of equal to or at least about 10 mPa ⁇ s at 20 °C at a concentration of 5% w/w in water.
  • Formula I has the stereochemical configuration shown in Formula I’:
  • the at least one of X 1 , X 2 , and X 3 is represented by the following:
  • the R 1 is -CH 2 C(O)-X 1 -((CH 2 ) g X 2 ) h ((CH 2 ) j X 3 )k- (CH 2 ) 1 H.
  • R 2 and R 3 are -OH.
  • each instance of R 1 , R 2 , and R 3 is independently selected from the group consisting of:
  • the compound has a viscosity of equal to or at least about 20 mPa ⁇ s at 20 °C at a concentration of 20% w/w in water. In several embodiments, the compound is soluble in water at a concentration of 50 mg/ml. In several embodiments, the compound has a total releasable NO storage in a range of 0.1-1.0 ⁇ mol of NO per mg of compound. In several embodiments, the compound has a NO half-life in the range of 0.1– 24 hours. In several embodiments, the compound has a total duration of NO release in the range of 1-60 hours. In several embodiments, the total NO release after 4 hours is in the range between 0.1-1.0 ⁇ mol of NO per mg of compound.
  • more than 15% of the repeat units in the compound are monomers of Formula I.
  • the compound has a molecular weight in the range of about 90 kDa and about 700 kDa.
  • the compound comprises two or more different covalently modified monomers of Formula I.
  • an NO releasing hyaluronic acid- derived polymer compound comprising a unit structure of Formula II:
  • each instance of R 1 , R 2 , R 3 , R 4 , R 5 , and R6 is independently -OH, -NH 2 , -CH 2 OH, -C(O)OH, -NHC(O)-CH 3 , -O-((CH 2 ) a O) b -H, -O-((CH 2)aO) b -(CH 2 )cH, -O-(C 1 -5alkyl), -NH-((CH 2 )dNH)e-H, -NH-((CH 2 )dNH)e-(CH 2 ) f H, -X 1 -((C H 2 ) g X 2 ) h -H, -X 1 -((CH 2 ) g X 2 ) h -(CH 2 ) 1 H, or -X 1 -((CH 2 )gX 2 ) h ((CH 2 )
  • each instance of a, b, c, d, e, f, g, h, i, j, k, and l is independently selected from an integer of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • each instance of X 1 , X 2 , and X 3 is independently selected from -O-, -S-, -NH-, C(O)NH-.
  • at least one of X 1 , X 2 , and X 3 is represented by one of the following:
  • the compound has a viscosity of equal to or at least about 10 mPa ⁇ s at 20 °C at a concentration of 5% w/w in water.
  • Formula II has the stereochemical configuration shown in Formula II’:
  • At least one of X 1 , X 2 , and X 3 is represented by the following:
  • R 1 is -CH 2 C(O)-X 1 -((CH 2 ) g X 2 ) h ((CH 2 ) j X 3 ) k - (CH 2 ) 1 H.
  • R 2 and R 3 are -OH.
  • one or more of R 1 , R 2 , R 3 , R 4 , R 5 , and R6 are independently selected from the group consisting of:
  • the compound has a viscosity of equal to or at least about 20 mPa ⁇ s at 20 °C at a concentration of 20% w/w in water. In several embodiments, the compound is soluble in water at a concentration of 50 mg/ml. In several embodiments, the compound has a total releasable NO storage in a range of 0.1-1.0 ⁇ mol of NO per mg of compound. In several embodiments, the compound has a NO half-life in the range of 0.1– 24 hours. In several embodiments, the compound has a total duration of NO release in the range of 1-60 hours. In several embodiments, the total NO release after 4 hours is in the range between 0.1-1.0 ⁇ mol of NO per mg of compound. In several embodiments, the compound has a molecular weight in the range of about 6 kDa and about 90 kDa.
  • the polymer comprises a polyaminoglycoside.
  • the polyaminoglycoside is a hyperbranched polyaminoglycoside, comprising a first aminoglycoside of Formula III:
  • G 1 is selected from the group consisting of:
  • G 2 is selected from the group consisting of:
  • each instance of R 1 is independently selected from the group consisting of -H, optionally substituted C 1 -C 6 alkyl, optionally substituted polyamino having 1 to 6 repeat units with intervening C 1 -C 6 alkyl groups, optionally substituted polyether having 1 to 6 repeat units with intervening C 1 -C 6 alkyl groups, or indicates a covalent bond to a linking unit.
  • each instance of X a is independently selected from -H, -OH, and C 1 -C 6 alkyl.
  • at least one instance of R 1 indicates a covalent bond to one or more linking unit selected from the following:
  • each instance of W 1 is independently selected from one or more additional aminoglycosides or one or more end-capping substituents and at least one linking unit provides a covalent bridge from the first aminoglycoside to a second aminoglycoside.
  • each instance of R a is independently selected from the group consisting of optionally substituted C 1 -C 6 alkyl, optionally substituted polyamino having 1 to 6 repeat units (with C 1 -C 6 alkyl(s)), or optionally substituted polyether having 1 to 6 repeat units (with C 1 -C 6 alkyl(s)).
  • the one or more end-capping substituents where present, independently has a formula of -X 1 -((CH 2 ) h X 2 )i-(CH 2 ) j H.
  • the polymer further comprises an end group selected from the group consisting of:
  • the polymer comprises an end group selected from the group consisting of:
  • Several embodiments pertain to a method of delivering nitric oxide to a subject in need of treatment.
  • an effective amount of the compounds or viscosity inducing agents is administered to the subject.
  • the effective amount of the compounds or viscosity inducing agents is administered as a hydrogel or the hydrogel is formed at the site of administration (e.g., in vivo).
  • the subject is a patient who has suffered a wound and the compounds or viscosity inducing agents are administered to aid in wound healing.
  • the subject is in need of tissue replacement and the compounds or viscosity inducing agents are administered as a tissue scaffold or filler and/or tissue re-growth promoting agents.
  • a method of treating a disease state an effective amount of the compounds or viscosity inducing agents is administered to a subject in need thereof, wherein said disease state is selected from the group consisting of a cancer, a cardiovascular disease, a microbial infection, platelet aggregation and platelet adhesion caused by the exposure of blood to a medical device, pathological conditions resulting from abnormal cell proliferation, transplantation rejections, autoimmune diseases, inflammation, vascular diseases, scar tissue, wound contraction, restenosis, pain, fever, gastrointestinal disorders, respiratory disorders, sexual dysfunctions, and sexually transmitted diseases.
  • Some embodiments pertain to a pharmaceutical formulation comprising the compounds (e.g., polymers) or viscosity inducing agents and a pharmaceutically acceptable excipient.
  • Some embodiments pertain to a method of reducing or preventing microbial load on a surface.
  • the compounds or viscosity inducing agents are applied to a surface contaminated with a plurality of microbes.
  • the compounds or viscosity generate nitric oxide and induce oxidative and/or nitrosative damage to microbial DNA and membrane structures, thereby preventing or reducing microbial load.
  • the plurality of microbes comprises one, two, or more of the following: gram-positive bacteria, gram-negative bacteria, fungi, yeast, and viruses.
  • the surface is an organic surface. In several embodiments, the surface is human skin.
  • the surface is an epithelial tissue. In several embodiments, the surface is a wound surface. In several embodiments, the surface is animal skin. In several embodiments, the application does not induce skin irritation. [0035] In several embodiments, the surface is an inorganic surface. In several embodiments, the inorganic surface is an external or internal surface of a medical device. In several embodiments, the application of the compound generates an anti-microbial coating on the external or internal surface of the medical device. In several embodiments, the medical device comprises an endoscope.
  • the microbial load comprises drug-resistant bacteria.
  • the microbial load comprises microbes associated with the presence of one or more of human immunodeficiency virus, herpes simplex virus, papilloma virus, parainfluenza virus, influenza, hepatitis, Coxsackie Virus, herpes zoster, measles, mumps, rubella, rabies, pneumonia, hemorrhagic viral fevers, H1N1, prions, parasites, fungi, mold, Candida albicans, Aspergillus niger, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, Group A streptococci, S.
  • the microbial load comprises Methicillin- resistant Staphylococcus aureus. In several embodiments, the microbial load comprises carbapenem-resistant Enterobacteriaceae. In several embodiments, the microbial load comprises Staphylococcus aureus. In several embodiments, the microbial load comprises Pseudomonas aeruginosa. In several embodiments, the microbial load comprises Burkholderia cepacia.
  • the NO donor generates nitric oxide and induces damage to the membrane and/or DNA of the microbes, thereby reducing the number of viable microbes.
  • the plurality of microbes comprises one or more of viruses, gram positive bacteria, gram negative bacteria, drug resistant bacteria, molds, yeasts, fungi, and combinations thereof.
  • the method comprises contacting a surface contaminated with a plurality of microbes (or that a surface that could be exposed to microbes) with a NO-releasing scaffold.
  • a NO-donor of the scaffold generates NO and induces damage to the membrane and/or DNA of the microbes, thereby reducing the number of viable microbes and/or preventing the colonization or infection of an area with microbes.
  • the surface comprises an organic surface. In some embodiments of the method, the surface is human skin or animal skin.
  • the surface is in the mouth, or surrounding tissues (e.g., lips, nasal nares, teeth, gums, etc.). In several embodiments, the surface comprises the oral mucosa. In some embodiments, the surface is in the lungs. In some embodiments, the surface is an inorganic surface (of a device, etc.). In several embodiments, the surface is an inorganic surface. In several embodiments, the inorganic surface is an external or internal surface of a medical device. In several embodiments, the device is a dental device.
  • the application step does not induce skin or tissue irritation.
  • the plurality of microbes comprises one or more of viruses, gram positive bacteria, gram negative bacteria, drug resistant bacteria, molds, yeasts, fungi, and combinations thereof.
  • Several embodiments pertain to a method of manufacturing any one of the compounds or viscosity inducing agents disclosed herein, comprising selecting a polymer and functionalizing the polymer with a NO binding moiety.
  • the polymer is a biopolymer.
  • the method includes exposing the compounds or viscosity inducing agents to NO to provide a NO donating compound or viscosity inducing agent.
  • compositions and related methods set forth in further detail below describe certain actions taken by a practitioner; however, it should be understood that they can also include the instructions of those actions by another party.
  • actions such as “administering a NO-donating scaffold” include“instructing the administration of a NO- donating scaffold.”
  • Figure 1 shows FTIR analysis of amine-modified CMC.
  • Figure 2 shows purity analysis of amine-functionalized hyaluronic acid derivatives.
  • A shows analysis of unreacted starting materials
  • B shows analysis of amine- modified 6 kDa HA derivatives
  • C shows analysis of amine-modified 90 kDa HA derivatives via HPLC-ELSD.
  • the amine-modified HA derivatives contain no detectable amounts of EDC and NHS reactants.
  • Figure 3 shows representative 1 H NMR and 13 C NMR spectra of unmodified hyaluronic acid.
  • (A) shows representative 1 H NMR and
  • (B) shows representative 13 C NMR spectra of HA6 in D 2 O.
  • Figure 4 shows representative 1 H NMR spectra of secondary amine- functionalized hyaluronic acid.
  • A shows representative 1 H NMR of HA6-PAPA
  • B shows representative 1 H NMR of HA90-PAPA
  • C shows representative 1 H NMR of HA6- HEDA
  • D shows representative 1 H NMR of HA90-HEDA
  • E shows representative 1 H NMR of HA6-DPTA
  • F shows representative 1 H NMR of HA90-DPTA
  • G shows representative 1 H NMR of HA6-DETA
  • H shows representative 1 H NMR of HA90- DETA, in D2O.
  • Figure 5 shows representative 13 C NMR spectra for secondary amine- functionalized hyaluronic acid, and a comparison of representative 13 C NMR for unmodified and amine-modified hyaluronic acid.
  • A shows representative 13 C NMR of HA6-PAPA
  • B shows representative 13 C NMR of HA90-PAPA
  • C shows representative 13 C NMR of HA6-HEDA
  • D shows representative 13 C NMR of HA90-HEDA
  • E shows representative 13 C NMR of HA6-DPTA
  • F shows representative 13 C NMR of HA90-DPTA
  • G shows representative 13 C NMR of HA6-DETA
  • H shows representative 13 C NMR of HA90- DETA, in D 2 O.
  • I shows representative 13 C NMR of unmodified and amine-modified hyaluronic acid.
  • Figure 6 shows representative UV-Vis spectra for the following secondary amine-functionalized and NO-releasing hyaluronic acid: (A) HA6-PAPA, (B) HA90- PAPA, (C) HA6-HEDA, (D) HA90-HEDA, (E) HA6-DPTA, (F) HA90-DPTA, (G) HA6- DETA, and (H) HA90-DETA. Modifications include: representative UV-Vis spectra of control (––) and NO-releasing (- -) hyaluronic acid.
  • Figure 7 shows real-time NO-release profiles and cumulative NO release of 6 and 90 kDa NO-releasing hyaluronic acid.
  • A-B shows real-time NO-release profiles for the initial 30 minutes of release and
  • C-D cumulative NO-release totals for (A, C) 6 kDa and (B, D) 90 kDa NO-releasing hyaluronic acid in PBS (10 mM, pH 7.4, 37 °C), wherein modifications include PAPA (––), HEDA (––), DPTA (•••), and DETA (–•–).
  • E also shows real-time measurement of NO release.
  • Figure 8 shows FTIR spectra of unmodified and amine-modified CMC scaffolds.
  • Figure 9 shows time-based bactericidal assay of 6 kDa NO-releasing HA derivatives against (A) P. aeruginosa and (B) S. aureus.
  • Treatments include HA6- PAPA/NO (blue circle), HA6-HEDA/NO (green square), HA6-DPTA/NO (red triangle), HA6-DETA/NO (purple diamond), and untreated (black cross). All HA derivatives were prepared at equivalent doses of 2 mg mL -1 for P. aeruginosa eradication and 16 mg mL -1 for S. aureus eradication.
  • Figure 10 shows antibacterial efficacy of active ingredient (neomycin or NO) against (A) E. coli, (B) P. aeruginosa, (C) S. aureus, (D) E. faecalis, (E) MDR-P. aeruginosa, and (F) MRSA following treatment with HA6-DPTA/NO (blue circle), HA90- DPTA/NO (green square), or neomycin sulfate (red triangle).
  • the NO dose was calculated from the total NO released over the 4 h exposure time in PBS (10 mM, pH 7.4, 37 °C) for HA6-DPTA/NO and HA90-DPTA/NO.
  • Figure 11 shows biofilm viability following 24 h treatment of (A) P. aeruginosa and (B) MDR-P. aeruginosa pre-existing biofilms in solution with equivalent active ingredient doses of neomycin sulfate (solid) or HA6-DPTA/NO (striped).
  • Figure 12 shows in vitro cytotoxicity results.
  • A1 shows concentration of amine-modified (solid) and NO-releasing (striped) hyaluronic acid derivatives required to reduce enzymatic activity of L929 murine fibroblasts by 50% (IC50).
  • A2) similarly shows concentration of amine-modified and NO-releasing hyaluronic acid derivatives required to reduce enzymatic activity of L929 murine fibroblasts by 50% (IC 50 ), as well as the inhibitory active ingredient dose of the NO-releasing hyaluronic acid.
  • HA6 is the left bar of each pair of bars.
  • B shows dose of NO released from hyaluronic acid derivatives required to reduce enzymatic activity by 50%.
  • Figure 13 shows antibacterial efficacy of 6 and 90 kDa NO-releasing hyaluronic acid derivatives against E. coli, P. aeruginosa, S. aureus, and E. faecalis.
  • Antibacterial efficacy of (A-D) 6 kDa and (E-H) 90 kDa NO-releasing hyaluronic acid against (A, E) E. coli, (B, F) P. aeruginosa, (C, G) S. aureus, and (D, H) E. faecalis is shown. Modifications include PAPA (blue circle), HEDA (green square), DPTA (red triangle), and DETA (purple diamond).
  • Figure 14 shows colonies remaining following treatment of E. coli, P. aeruginosa, S. aureus, and E. faecalis with 6 and 90 kDa amine-functionalized (control) hyaluronic acid.
  • Figure 15 shows bacteria killing curves for 6 and 90 kDa DPTA- functionalized and NO-releasing hyaluronic acid against MDR-P. aeruginosa and MRSA. Antibacterial efficacy of 6 kDa (circle) and 90 kDa (square) DPTA-modified (hollow) and NO-releasing (solid) hyaluronic acid against antibiotic-resistant bacteria strains, including multidrug-resistant P. aeruginosa (red) and methicillin-resistant S. aureus (blue) is shown.
  • Figure 16 shows antibiofilm efficacy of 6 and 90 kDa NO-releasing DPTA- modified HA against P. aeruginosa and MDR-P. aeruginosa biofilms.
  • Biofilm viability following 24 h treatment of (A-B) P. aeruginosa and (C-D) MDR-P. aeruginosa biofilms with HA6-DPTA/NO (blue circle), HA90-DPTA/NO (green square), or neomycin sulfate (red triangle) is shown.
  • Another figuration of the biofilm viability of 6 and 90 kDa NO- releasing DPTA-modified HA and neomycin against P. aeruginosa and MDR-P. aeruginosa biofilms is shown in (E) and (F).
  • Figure 17 shows biofilm viability following 24 h exposure of P. aeruginosa and MDR-P. aeruginosa biofilms to blank (left, gray), control HA6-DPTA (middle, blue), or control HA90-DPTA (right, green) solutions. Solutions of HA were prepared at the MBEC 24h for the respective NO-releasing derivative. Of note, HA90-DPTA was prepared at 32 mg mL -1 for testing of MDR-P. aeruginosa biofilms due to the lack of MBEC24h for the NO-releasing derivative.
  • Figure 18 shows viability of L929 murine fibroblasts following 24 h treatment with unmodified 6 kDa (blue, left) and 90 kDa (green, right) hyaluronic acid.
  • Figure 19 shows dose-response curves after 24 h treatment of L929 murine fibroblasts with amine-modified (hollow) and NO-releasing (solid) HA derivatives.
  • Modifications of 6 kDa (red circle) and 90 kDa (blue square) HA include (A) PAPA, (B) HEDA, (C) DPTA, and (D) DETA.
  • Figure 20 shows metabolic activity of HGF-1 cells determined via MTS assay after 24 h exposure to (A) CMC-DETA (solid), CMC-DPTA (diagonal stripes), CMC- HEDA (horizontal stripes), and CMC-PAPA (dotted) and (B) CMC-DETA/NO (solid), CMC-DPTA/NO (diagonal stripes), CMC-HEDA/NO (horizontal stripes), and CMC- PAPA/NO (dotted).
  • Figure 21 shows UV-vis absorption spectra of A) CMC-DETA (solid line) and CMC-DETA/NO (dashed line), B) CMC-DPTA (solid line) and CMC-DPTA/NO (dashed line), C) CMC-HEDA (solid line) and CMC-HEDA/NO (dashed line), and D) CMC-PAPA (solid line) and CMC-PAPA/NO (dashed line), in 50 mM NaOH.
  • the scaffolds comprise polymers.
  • the scaffolds comprise biopolymers.
  • the scaffolds comprise biocompatible polymers.
  • the scaffolds comprise one or more saccharide units and/or are polysaccharides.
  • the scaffolds comprise one or more chitosan, hyaluronic acid (HA), carboxymethylcellulose (CMC), hydroxyethyl cellulose, methyl cellulose, cellulose, alginate (including 1,4-linked a-1-guluronic acid (G) and b-d-mannuronic acid (M) units), collagen, gelatin, cyclodextrin (e.g., having 5 (a), 6 (b), 7 (g), or more a-D-glucopyranosides), aminoglycosides (e.g., kanamycin, streptomycin, tobramycin, gentamicin, neomycin, etc.), elastin, repeat units thereof, structural units thereof, or combinations thereof.
  • HA hyaluronic acid
  • CMC carboxymethylcellulose
  • M b-d-mannuronic acid
  • collagen gelatin
  • cyclodextrin e.g., having 5 (a), 6 (b), 7 (g), or more a
  • one or more polymers are crosslinked to form the scaffold.
  • the polymers are not crosslinked to form the scaffold.
  • the scaffolds allow the efficient reduction in viability and/or eradication of microbes (e.g., prokaryotic cells, bacteria, protozoa, fungi, algae, amoebas, slime molds, etc. and in particular such microbes that have developed at least some degree of drug resistance) with low toxicity native tissue and patient cells (e.g., eukaryotic cells, mammalian cells, human cells, etc.).
  • microbes e.g., prokaryotic cells, bacteria, protozoa, fungi, algae, amoebas, slime molds, etc. and in particular such microbes that have developed at least some degree of drug resistance
  • native tissue and patient cells e.g., eukaryotic cells, mammalian cells, human cells, etc.
  • the term“effective amount,” as used herein, refers to that amount of a recited compound that imparts a modulating effect, which, for example, can be a beneficial effect, to a subject afflicted with a disorder, disease or illness, including improvement in the condition of the subject (e.g., in one or more symptoms), delay or reduction in the progression of the condition, prevention or delay of the onset of the disorder, and/or change in clinical parameters, disease or illness, etc., as would be well known in the art.
  • an effective amount can refer to the amount of a composition, compound, or agent that improves a condition in a subject by at least 5%, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.
  • an improvement in a condition can be a reduction in infection.
  • an improvement can be reduction of bacterial load (e.g., bioburden) on a surface or in a subject.
  • Actual dosage levels of active ingredients in an active composition of the presently disclosed subject matter can be varied so as to administer an amount of the active compound(s) that is effective to achieve the desired response for a particular subject and/or application.
  • the selected dosage level will depend upon a variety of factors including, but not limited to, the activity of the composition, formulation, route of administration, combination with other drugs or treatments, severity of the condition being treated, and the physical condition and prior medical history of the subject being treated.
  • a minimal dose is administered, and dose is escalated in the absence of dose-limiting toxicity to a minimally effective amount. Determination and adjustment of an effective dose, as well as evaluation of when and how to make such adjustments, are contemplated herein.
  • biopolymer refers to a polymeric substance occurring in living organisms, including polynucleotides (e.g., DNA, RNA), polysaccharides (e.g., cellulose), proteins (e.g., polypeptides), glycopeptides, peptidoglycans, and the like.
  • polynucleotides e.g., DNA, RNA
  • polysaccharides e.g., cellulose
  • proteins e.g., polypeptides
  • glycopeptides e.g., glycopeptides, peptidoglycans, and the like.
  • “Treat” or“treating” or“treatment” refers to any type of action that imparts a modulating effect, which, for example, can be a beneficial effect, to a subject afflicted with a disorder, disease or illness, including improvement in the condition of the subject (e.g., in one or more symptoms), delay or reduction in the progression of the condition, and/or change in clinical parameters, disease or illness, curing the illness, etc.
  • the terms“disrupting” and“eradicating” refer to the ability of the presently disclosed structures to combat biofilms.
  • the biofilms may be partially eradicated or disrupted, meaning that the cells no longer attach to one another or to a surface.
  • the biofilm may be completely eradicated, meaning that the biofilm is no longer an interconnected, cohesive, or continuous network of cells to a substantial degree.
  • nitric oxide donor or“NO donor” refer to species and/or molecules that donate, release and/or directly or indirectly transfer a nitric oxide species, and/or stimulate the endogenous production of nitric oxide in vivo and/or elevate endogenous levels of nitric oxide in vivo such that the biological activity of the nitric oxide species is expressed at the intended site of action.
  • the terms“nitric oxide releasing” or“nitric oxide donating” refer to species that donate, release and/or directly or indirectly transfer any one (or two or more) of the three redox forms of nitrogen monoxide (NO+, NO-, NO (e.g., *NO)) and/or methods of donating, releasing and/or directly or indirectly transferring any one (or two or more) of the three redox forms of nitrogen monoxide (NO+, NO-, NO).
  • the nitric oxide releasing is accomplished such that the biological activity of the nitrogen monoxide species is expressed at the intended site of action.
  • microbial infection refers to bacterial, fungal, viral, yeast infections, as well other microorganisms, and combinations thereof.
  • the “patient” or “subject” treated as disclosed herein is, in some embodiments, a human patient, although it is to be understood that the principles of the presently disclosed subject matter indicate that the presently disclosed subject matter is effective with respect to all vertebrate species, including mammals, which are intended to be included in the terms“subject” and“patient.” Suitable subjects are generally mammalian subjects. The subject matter described herein finds use in research as well as veterinary and medical applications.
  • the term“mammal” as used herein includes, but is not limited to, humans, non-human primates, cattle, sheep, goats, pigs, horses, cats, dog, rabbits, rodents (e.g., rats or mice), monkeys, etc.
  • Human subjects include neonates, infants, juveniles, adults and geriatric subjects.
  • the subject“in need of’ the methods disclosed herein can be a subject that is experiencing a disease state and/or is anticipated to experience a disease state, and the methods and compositions of the invention are used for therapeutic and/or prophylactic treatment.
  • Such physical properties include solubility, charge, stability, cross-linking, secondary and tertiary structure, and the like. Moreover, if no stereochemistry is indicated for compounds having one or more chiral centers, all enantiomers and diasteromers are included. Similarly, for a recitation of aliphatic or alkyl groups, all structural isomers thereof also are included.
  • groups shown as Ai through A n and referred to herein as an alkyl group are independently selected from alkyl or aliphatic groups, particularly alkyl having 20 or fewer carbon atoms, and even more typically lower alkyl having 10 or fewer atoms, such as methyl, ethyl, propyl, isopropyl, and butyl.
  • the alkyl may be optionally substituted (e.g., substituted or not substituted, as disclosed elsewhere herein).
  • the alkyl may be a substituted alkyl group, such as alkyl halide (e.g.— CX3 where X is a halide, and combinations thereof, either in the chain or bonded thereto,), alcohols (i.e. aliphatic or alkyl hydroxyl, particularly lower alkyl hydroxyl) or other similarly substituted moieties such as amino-, amino acid-, aryl-, alkyl aryl-, alkyl ester-, ether-, keto-, nitro-, sulfhydryl-, sulfonyl-, sulfoxide modified- alkyl groups.
  • alkyl halide e.g.— CX3 where X is a halide, and combinations thereof, either in the chain or bonded thereto
  • alcohols i.e. aliphatic or alkyl hydroxyl, particularly lower alkyl hydroxyl
  • moieties such as amino-, amino acid-, aryl-, alkyl
  • amino and“amine” refer to nitrogen-containing groups such as NR 3 , NH 3 , NHR 2 , and NH 2 R, wherein R can be as described elsewhere herein.
  • amino as used herein can refer to a primary amine, a secondary amine, or a tertiary amine.
  • one R of an amino group can be a diazeniumdiolate (i.e., NONO).
  • the indicated “optionally substituted” or“substituted” group may be substituted with one or more group(s) individually and independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, aryl(alkyl), cycloalkyl(alkyl), heteroaryl(alkyl), heterocyclyl(alkyl), hydroxy, alkoxy, acyl, cyano, halogen, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, nitro, sulfenyl, sulfinyl, sulfonyl,
  • “C a to C b ” in which“a” and“b” are integers refer to the number of carbon atoms in a group.
  • the indicated group can contain from“a” to“b”, inclusive, carbon atoms.
  • a“C 1 to C 4 alkyl” or“C 1 - C 4 alkyl” group refers to all alkyl groups having from 1 to 4 carbons, that is, CH 3 -, CH 3 CH 2 -, CH 3 CH 2 CH 2 -, (CH 3 ) 2 CH-, CH 3 CH 2 CH 2 CH 2 -, CH 3 CH 2 CH(CH 3 )- and (CH 3 ) 3 C-. If no“a” and“b” are designated, the broadest range described in these definitions is to be assumed.
  • R groups and the atoms they are attached to can form a cycloalkyl, cycloalkenyl, aryl, heteroaryl or heterocycle.
  • R a and R b of an NR a R b group are indicated to be“taken together,” it means that they are covalently bonded to one another to form a ring:
  • alkyl refers to a fully saturated aliphatic hydrocarbon group.
  • the alkyl moiety may be branched or straight chain.
  • branched alkyl groups include, but are not limited to, iso-propyl, sec-butyl, t-butyl and the like.
  • straight chain alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl and the like.
  • the alkyl group may have 1 to 30 carbon atoms (whenever it appears herein, a numerical range such as“1 to 30” refers to each integer in the given range; e.g.,“1 to 30 carbon atoms” means that the alkyl group may consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon atoms, although the present definition also covers the occurrence of the term“alkyl” where no numerical range is designated).
  • The“alkyl” group may also be a medium size alkyl having 1 to 12 carbon atoms.
  • The“alkyl” group could also be a lower alkyl having 1 to 6 carbon atoms.
  • alkyl group may be substituted or unsubstituted.
  • “C 1 -C 5 alkyl” indicates that there are one to five carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl (branched and straight-chained), etc.
  • Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl and hexyl.
  • alkylene refers to a bivalent fully saturated straight chain aliphatic hydrocarbon group.
  • alkylene groups include, but are not limited to, methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene and octylene.
  • An alkylene group may be represented by , followed by the number of carbon atoms, followed by a“*”. For example, to represent ethylene.
  • the alkylene group may have 1 to 30 carbon atoms (whenever it appears herein, a numerical range such as“1 to 30” refers to each integer in the given range; e.g.,“1 to 30 carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 30 carbon atoms, although the present definition also covers the occurrence of the term“alkylene” where no numerical range is designated).
  • the alkylene group may also be a medium size alkyl having 1 to 12 carbon atoms.
  • the alkylene group could also be a lower alkyl having 1 to 6 carbon atoms.
  • An alkylene group may be substituted or unsubstituted.
  • a lower alkylene group can be substituted by replacing one or more hydrogens of the lower alkylene group and/or by substituting both hydrogens on the same carbon with a C3-6 monocyclic cycloalkyl group (e.g., ).
  • alkenyl refers to a monovalent straight or branched chain radical of from two to twenty carbon atoms containing a carbon double bond(s) including, but not limited to, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2- butenyl and the like.
  • An alkenyl group may be unsubstituted or substituted.
  • alkynyl refers to a monovalent straight or branched chain radical of from two to twenty carbon atoms containing a carbon triple bond(s) including, but not limited to, 1-propynyl, 1-butynyl, 2-butynyl and the like.
  • An alkynyl group may be unsubstituted or substituted.
  • cycloalkyl refers to a completely saturated (no double or triple bonds) mono- or multi- cyclic (such as bicyclic) hydrocarbon ring system. When composed of two or more rings, the rings may be joined together in a fused, bridged or spiro fashion.
  • the term“fused” refers to two rings which have two atoms and one bond in common.
  • the term“bridged cycloalkyl” refers to compounds wherein the cycloalkyl contains a linkage of one or more atoms connecting non-adjacent atoms.
  • Cycloalkyl groups can contain 3 to 30 atoms in the ring(s), 3 to 20 atoms in the ring(s), 3 to 10 atoms in the ring(s), 3 to 8 atoms in the ring(s) or 3 to 6 atoms in the ring(s).
  • a cycloalkyl group may be unsubstituted or substituted.
  • Examples of mono-cycloalkyl groups include, but are in no way limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.
  • Examples of fused cycloalkyl groups are decahydronaphthalenyl, dodecahydro-1H-phenalenyl and tetradecahydroanthracenyl;
  • examples of bridged cycloalkyl groups are bicyclo[1.1.1]pentyl, adamantanyl and norbornanyl; and examples of spiro cycloalkyl groups include spiro[3.3]heptane and spiro[4.5]decane.
  • cycloalkenyl refers to a mono- or multi- cyclic (such as bicyclic) hydrocarbon ring system that contains one or more double bonds in at least one ring; although, if there is more than one, the double bonds cannot form a fully delocalized pi-electron system throughout all the rings (otherwise the group would be“aryl,” as defined herein).
  • Cycloalkenyl groups can contain 3 to 10 atoms in the ring(s), 3 to 8 atoms in the ring(s) or 3 to 6 atoms in the ring(s). When composed of two or more rings, the rings may be connected together in a fused, bridged, or spiro fashion.
  • a cycloalkenyl group may be unsubstituted or substituted.
  • aryl refers to a carbocyclic (all carbon) monocyclic or multicyclic (such as bicyclic) aromatic ring system (including fused ring systems where two carbocyclic rings share a chemical bond) that has a fully delocalized pi-electron system throughout all the rings.
  • the number of carbon atoms in an aryl group can vary.
  • the aryl group can be a C 6 -C 14 aryl group, a C 6 -C 10 aryl group or a C 6 aryl group.
  • Examples of aryl groups include, but are not limited to, benzene, naphthalene and azulene.
  • An aryl group may be substituted or unsubstituted.
  • heteroaryl refers to a monocyclic or multicyclic (such as bicyclic) aromatic ring system (a ring system with fully delocalized pi-electron system) that contain(s) one or more heteroatoms (for example, 1, 2 or 3 heteroatoms), that is, an element other than carbon, including but not limited to, nitrogen, oxygen and sulfur.
  • heteroatoms for example, 1, 2 or 3 heteroatoms
  • the number of atoms in the ring(s) of a heteroaryl group can vary.
  • the heteroaryl group can contain 4 to 14 atoms in the ring(s), 5 to 10 atoms in the ring(s) or 5 to 6 atoms in the ring(s), such as nine carbon atoms and one heteroatom; eight carbon atoms and two heteroatoms; seven carbon atoms and three heteroatoms; eight carbon atoms and one heteroatom; seven carbon atoms and two heteroatoms; six carbon atoms and three heteroatoms; five carbon atoms and four heteroatoms; five carbon atoms and one heteroatom; four carbon atoms and two heteroatoms; three carbon atoms and three heteroatoms; four carbon atoms and one heteroatom; three carbon atoms and two heteroatoms; or two carbon atoms and three heteroatoms.
  • heteroaryl includes fused ring systems where two rings, such as at least one aryl ring and at least one heteroaryl ring or at least two heteroaryl rings, share at least one chemical bond.
  • heteroaryl rings include, but are not limited to, furan, furazan, thiophene, benzothiophene, phthalazine, pyrrole, oxazole, benzoxazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, thiazole, 1,2,3-thiadiazole, 1,2,4- thiadiazole, benzothiazole, imidazole, benzimidazole, indole, indazole, pyrazole, benzopyrazole, isoxazole, benzoisoxazole, isothiazole, triazole, benzotriazole, thiadiazole, tetrazole, pyridine, pyridazine, pyrimidine
  • heteroaryl group may be substituted or unsubstituted.
  • “heterocyclyl” or“heteroalicyclyl” refers to three-, four-, five-, six-, seven-, eight-, nine-, ten-, up to 18-membered monocyclic, bicyclic and tricyclic ring system wherein carbon atoms together with from 1 to 5 heteroatoms constitute said ring system.
  • a heterocycle may optionally contain one or more unsaturated bonds situated in such a way, however, that a fully delocalized pi-electron system does not occur throughout all the rings.
  • the heteroatom(s) is an element other than carbon including, but not limited to, oxygen, sulfur and nitrogen.
  • a heterocycle may further contain one or more carbonyl or thiocarbonyl functionalities, so as to make the definition include oxo-systems and thio- systems such as lactams, lactones, cyclic imides, cyclic thioimides and cyclic carbamates.
  • the rings When composed of two or more rings, the rings may be joined together in a fused, bridged or spiro fashion.
  • the term“fused” refers to two rings which have two atoms and one bond in common.
  • bridged heterocyclyl or“bridged heteroalicyclyl” refers to compounds wherein the heterocyclyl or heteroalicyclyl contains a linkage of one or more atoms connecting non-adjacent atoms.
  • spiro refers to two rings which have one atom in common and the two rings are not linked by a bridge.
  • Heterocyclyl and heteroalicyclyl groups can contain 3 to 30 atoms in the ring(s), 3 to 20 atoms in the ring(s), 3 to 10 atoms in the ring(s), 3 to 8 atoms in the ring(s) or 3 to 6 atoms in the ring(s).
  • any nitrogens in a heteroalicyclic may be quaternized.
  • Heterocyclyl or heteroalicyclic groups may be unsubstituted or substituted.
  • heterocyclyl or“heteroalicyclyl” groups include but are not limited to, 1,3-dioxin, 1,3-dioxane, 1,4-dioxane, 1,2-dioxolane, 1,3- dioxolane, 1,4-dioxolane, 1,3-oxathiane, 1,4-oxathiin, 1,3-oxathiolane, 1,3-dithiole, 1,3- dithiolane, 1,4-oxathiane, tetrahydro-1,4-thiazine, 2H-1,2-oxazine, maleimide, succinimide, barbituric acid, thiobarbituric acid, dioxopiperazine, hydantoin, dihydrouracil, trioxane, hexahydro-1,3,5-triazine, imidazoline, imidazolidine, isoxazoline, isoxazolidine,
  • spiro heterocyclyl groups examples include 2- azaspiro[3.3]heptane, 2-oxaspiro[3.3]heptane, 2-oxa-6-azaspiro[3.3]heptane, 2,6- diazaspiro[3.3]heptane, 2-oxaspiro[3.4]octane and 2-azaspiro[3.4]octane.
  • aralkyl and“aryl(alkyl)” refer to an aryl group connected, as a substituent, via a lower alkylene group.
  • the lower alkylene and aryl group of an aralkyl may be substituted or unsubstituted. Examples include but are not limited to benzyl, 2- phenylalkyl, 3-phenylalkyl and naphthylalkyl.
  • cycloalkyl(alkyl) refer to an cycloalkyl group connected, as a substituent, via a lower alkylene group.
  • the lower alkylene and cycloalkyl group of a cycloalkyl(alkyl) may be substituted or unsubstituted.
  • heteroaryl and“heteroaryl(alkyl)” refer to a heteroaryl group connected, as a substituent, via a lower alkylene group.
  • the lower alkylene and heteroaryl group of heteroaralkyl may be substituted or unsubstituted. Examples include but are not limited to 2-thienylalkyl, 3-thienylalkyl, furylalkyl, thienylalkyl, pyrrolylalkyl, pyridylalkyl, isoxazolylalkyl and imidazolylalkyl and their benzo-fused analogs.
  • A“heteroalicyclyl(alkyl)” and“heterocyclyl(alkyl)” refer to a heterocyclic or a heteroalicyclic group connected, as a substituent, via a lower alkylene group.
  • the lower alkylene and heterocyclyl of a (heteroalicyclyl)alkyl may be substituted or unsubstituted. Examples include but are not limited tetrahydro-2H-pyran-4-yl(methyl), piperidin-4- yl(ethyl), piperidin-4-yl(propyl), tetrahydro-2H-thiopyran-4-yl(methyl) and 1,3-thiazinan- 4-yl(methyl).
  • alkoxy refers to the Formula–OR wherein R is an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl) is defined herein.
  • R is an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl) is defined herein.
  • a non-limiting list of alkoxys are methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, iso-butoxy, sec
  • acyl refers to a hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, aryl(alkyl), heteroaryl(alkyl) and heterocyclyl(alkyl) connected, as substituents, via a carbonyl group. Examples include formyl, acetyl, propanoyl, benzoyl and acryl. An acyl may be substituted or unsubstituted.
  • a“cyano” group refers to a“-CN” group.
  • halogen atom or“halogen” as used herein, means any one of the radio-stable atoms of column 7 of the Periodic Table of the Elements, such as, fluorine, chlorine, bromine and iodine.
  • a thiocarbonyl may be substituted or unsubstituted.
  • An O-carbamyl may be substituted or unsubstituted.
  • An N-carbamyl may be substituted or unsubstituted.
  • An O-thiocarbamyl may be substituted or unsubstituted.
  • An N-thiocarbamyl may be substituted or unsubstituted.
  • a C-amido may be substituted or unsubstituted.
  • An N-amido may be substituted or unsubstituted.
  • An“S-sulfonamido” group refers to a“-SO 2 N(R A R B )” group in which R A and R B can be independently hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl).
  • An S-sulfonamido may be substituted or unsubstituted.
  • An“N-sulfonamido” group refers to a“RSO 2 N(R A )-” group in which R and RA can be independently hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl).
  • An N-sulfonamido may be substituted or unsubstituted.
  • An O-carboxy may be substituted or unsubstituted.
  • An ester and C-carboxy may be substituted or unsubstituted.
  • A“nitro” group refers to an“–NO 2 ” group.
  • A“sulfenyl” group refers to an“-SR” group in which R can be hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl).
  • R can be hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl).
  • a sulfenyl may be substituted or unsubstituted.
  • a sulfinyl may be substituted or unsubstituted.
  • A“sulfonyl” group refers to an“SO 2 R” group in which R can be the same as defined with respect to sulfenyl.
  • a sulfonyl may be substituted or unsubstituted.
  • haloalkyl refers to an alkyl group in which one or more of the hydrogen atoms are replaced by a halogen (e.g., mono-haloalkyl, di-haloalkyl, tri- haloalkyl and polyhaloalkyl).
  • a halogen e.g., mono-haloalkyl, di-haloalkyl, tri- haloalkyl and polyhaloalkyl.
  • halogen e.g., mono-haloalkyl, di-haloalkyl, tri- haloalkyl and polyhaloalkyl.
  • Such groups include but are not limited to, chloromethyl, fluoromethyl, difluoromethyl, trifluoromethyl, 1-chloro-2-fluoromethyl, 2-fluoroisobutyl and pentafluoroethyl.
  • a haloalkyl may be substituted or unsubstituted.
  • haloalkoxy refers to an alkoxy group in which one or more of the hydrogen atoms are replaced by a halogen (e.g., mono-haloalkoxy, di- haloalkoxy and tri-haloalkoxy).
  • a halogen e.g., mono-haloalkoxy, di- haloalkoxy and tri-haloalkoxy.
  • groups include but are not limited to, chloromethoxy, fluoromethoxy, difluoromethoxy, trifluoromethoxy, 1-chloro-2-fluoromethoxy and 2- fluoroisobutoxy.
  • a haloalkoxy may be substituted or unsubstituted.
  • A“mono-substituted amine” group refers to a“-NHR A ” group in which R A can be an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl), as defined herein.
  • the RA may be substituted or unsubstituted.
  • a mono-substituted amine group can include, for example, a mono-alkylamine group, a mono-C 1 -C 6 alkylamine group, a mono-arylamine group, a mono-C 6 -C 10 arylamine group and the like.
  • Examples of mono-substituted amine groups include, but are not limited to, -NH(methyl), -NH(phenyl) and the like.
  • A“di-substituted amine” group refers to a“-NRAR B ” group in which RA and R B can be independently an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl (alkyl), as defined herein.
  • R A and R B can independently be substituted or unsubstituted.
  • a di-substituted amine group can include, for example, a di-alkylamine group, a di-C 1 -C 6 alkylamine group, a di-arylamine group, a di-C 6 -C 10 arylamine group and the like.
  • Examples of di-substituted amine groups include, but are not limited to, -N(methyl)2, -N(phenyl)(methyl), -N(ethyl)(methyl) and the like.
  • “mono-substituted amine(alkyl)” group refers to a mono-substituted amine as provided herein connected, as a substituent, via a lower alkylene group.
  • a mono-substituted amine(alkyl) may be substituted or unsubstituted.
  • a mono-substituted amine(alkyl) group can include, for example, a mono-alkylamine(alkyl) group, a mono-C 1 -C 6 alkylamine(C 1 -C 6 alkyl) group, a mono-arylamine(alkyl group), a mono-C 6 -C 10 arylamine(C 1 -C 6 alkyl) group and the like.
  • Examples of mono-substituted amine(alkyl) groups include, but are not limited to, -CH 2 NH(methyl), -CH 2 NH(phenyl), -CH 2 CH 2 NH(methyl), -CH 2 CH 2 NEXphenyl) and the like.
  • di-substituted amine(alkyl) refers to a di-substituted amine as provided herein connected, as a substituent, via a lower alkylene group.
  • a di-substituted amine(alkyl) may be substituted or unsubstituted.
  • a di-substituted amine(alkyl) group can include, for example, a dialkylamine(alkyl) group, a di-C 1 -C 6 alkylamine(C 1 -C 6 alkyl) group, a di-arylamine(alkyl) group, a di-C 6 -C 10 arylamine(C 1 -C 6 alkyl) group and the like.
  • di-substituted amine(alkyl)groups include, but are not limited to, -CH 2 N(methyl)2, -CH 2 N(phenyl)(methyl), -CH 2 N(ethyl)(methyl), -CH 2 CH 2 N(methyl)2, -CH 2 CH 2 N(phenylXmethyl), -NCH 2 CH 2 (ethylXmethyl) and the like.
  • the term“diamino-” denotes a“-N(RA)RB-N(RC)(RD)” group in which RA, RC, and R D can be independently a hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl), as defined herein, and wherein R B connects the two“N” groups and can be (independently of RA, RC, and RD) a substituted or unsubstituted alkylene group.
  • RA, RB, RC, and RD can independently further be substituted or unsubstituted.
  • polyamino denotes a“-(N(RA)RB-)n- N(RC)(RD)”.
  • polyamino can comprise -N(RA)alkyl-N(RA)alkyl- N(R A )alkyl-N(R A )alkyl-H.
  • the alkyl of the polyamino is as disclosed elsewhere herein. While this example has only 4 repeat units, the term“polyamino” may consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeat units.
  • RA, RC, and RD can be independently a hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl), as defined herein, and wherein RB connects the two“N” groups and can be (independently of R A , R C , and R D ) a substituted or unsubstituted alkylene group.
  • R A, R C , and R D can independently further be substituted or unsubstituted.
  • the polyamino comprises amine groups with intervening alkyl groups (where alkyl is as defined elsewhere herein).
  • the term“diether-” denotes an“-OR B O-R A ” group in which RA can be a hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl), as defined herein, and wherein R B connects the two“O” groups and can be a substituted or unsubstituted alkylene group.
  • R A can independently further be substituted or unsubstituted.
  • polyether denotes a repeating–(OR B -) n OR A group.
  • polyether can comprise -Oalkyl-Oalkyl-Oalkyl-Oalkyl- ORA.
  • the alkyl of the polyether is as disclosed elsewhere herein. While this example has only 4 repeat units, the term“polyether” may consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeat units.
  • R A can be a hydrogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkyl(alkyl), aryl(alkyl), heteroaryl(alkyl) or heterocyclyl(alkyl), as defined herein.
  • RB can be a substituted or unsubstituted alkylene group.
  • R A can independently further be substituted or unsubstituted.
  • the polyether comprises ether groups with intervening alkyl groups (where alkyl is as defined elsewhere herein and can be optionally substituted).
  • substituents there may be one or more substituents present.
  • “haloalkyl” may include one or more of the same or different halogens.
  • “C 1 -C 3 alkoxyphenyl” may include one or more of the same or different alkoxy groups containing one, two or three atoms.
  • a radical indicates species with a single, unpaired electron such that the species containing the radical can be covalently bonded to another species.
  • a radical is not necessarily a free radical. Rather, a radical indicates a specific portion of a larger molecule.
  • the term“radical” can be used interchangeably with the term“group.”
  • the range includes any number falling within the range and the numbers defining ends of the range.
  • integers included in the range are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc., up to and including 20.
  • the term“about,” as used herein when referring to a measurable value such as an amount of a compound or agent of this invention, dose, time, temperature, and the like, is meant to encompass variations of ⁇ 20%, ⁇ 10%, ⁇ 5%, ⁇ 1%, ⁇ 0.5%, or even ⁇ 0.1% of the specified amount.
  • the term“consists essentially of” (and grammatical variants), shall be given its ordinary meaning and shall also mean that the composition or method referred to can contain additional components as long as the additional components do not materially alter the composition or method.
  • the term “consists of” (and grammatical variants) shall be given its ordinary meaning and shall also mean that the composition or method referred to is closed to additional components.
  • the term“comprising” (and grammatical variants) shall be given its ordinary meaning and shall also mean that the composition or method referred to is open to contain additional components.
  • Nitric oxide an endogenously produced diatomic free radical
  • Deficiency of NO can lead to some degree of malfunction of NO-relevant physiological systems.
  • Exogenous NO delivery may be an effective strategy for the resolution of biomedical therapies ranging from cardiovascular diseases, to antibacterial and anticancer therapies. NO delivery can also be used to achieve antimicrobial activity.
  • N-diazeniumdiolates e.g., N-diazeniumdiolates, S-nitrosothiols, metal nitrosyls, organic nitrates
  • NONOates N-diazeniumdiolates
  • the NO donor comprises any one of the following nitric oxide releasing moieties:
  • the NO donor is a N-diazeniumdiolate NO donor.
  • the NO donor is attached along a linear unit at a secondary amine as disclosed elsewhere herein.
  • NO is also a potent antibacterial agent that acts on bacteria via nitrosative and/or oxidative stress.
  • NO is a broad- spectrum antibacterial agent and in some embodiments, scaffolds that deliver NO are capable of eradicating both bacteria and biofilms, primarily through the formation of reactive NO byproducts (e.g., peroxynitrite and dinitrogen trioxide) that cause oxidative and nitrosative damage to microbial DNA and/or membrane structures.
  • reactive NO byproducts e.g., peroxynitrite and dinitrogen trioxide
  • NO-releasing materials may be good targets to battle bacterial infection.
  • the antibacterial efficacy of NO-releasing materials may be dependent on both NO payloads and associated release kinetics.
  • high NO total is an important parameter to effectively evaluate storage capability of good scaffolds.
  • a high density of secondary amine groups imbues certain donors with a high NO storage capacity.
  • NO release that is too fast and high NO storage may result in undesired toxicity to mammalian cells. Therefore, challenges exist in preparing biocompatible NO-releasing materials with high NO storage and low cytotoxicity, and such challenges, among others, are addressed according to several embodiments disclosed herein.
  • Several embodiments disclosed herein have one or more of the following advantages: efficient and unique synthesis routes and resultant chemical composition of polymer constructs. Controllable amounts of secondary-amines and diverse exterior terminal groups (e.g., hydroxyl, methyl, hydroxymethyl, and primary amine) can be provided. The NO storage and NO-release kinetics of the generated nitric-oxide releasing scaffolds can be tuned for a particular application. This tuning is achieved, in several embodiments, by altering the type and/or number of functionalized monomers of the formulae disclosed herein.
  • additional functionalization of the amines in the generated nitric-oxide releasing scaffolds further enables the control over NO-release kinetics.
  • the secondary amine group directly influences the stability of the N- diazeniumdiolate (or other NO carrier group), allowing for control over both NO storage and release kinetics.
  • nitric oxide not only plays fundamental roles in several important biological processes, but also exhibits function as an antibacterial or anticancer agent.
  • various NO donors e.g., N- diazeniumdiolates, S-nitrosothiols, metal nitrosyls, organic nitrates
  • N-bound diazeniumdiolates are attractive because of their good stability and facile storage, which spontaneously undergo proton-triggered dissociation under physiological condition to regenerate the NO radicals.
  • biocompatible N- diazeniumdiolate-modified scaffolds including those derived from biopolymers and saccharide derived polymers (e.g., chitosan, hyaluronic acid, CMC, etc.).
  • NO an endogenously produced free radical, eradicates bacteria using a variety of mechanisms, including, but not limited to, lipid peroxidation, nitrosation of membrane proteins, and DNA damage via reactive oxygen/nitrogen species (e.g., peroxynitrite, dinitrogen trioxide).
  • reactive oxygen/nitrogen species e.g., peroxynitrite, dinitrogen trioxide.
  • NO has the improved ability to actively degrade both the biofilm matrix and mucus structure, thus allowing for more efficient biocidal action and mucociliary clearance.
  • the polymer scaffold is derived from a biopolymer.
  • the scaffold and/or biopolymer is water soluble.
  • the scaffold and/or biopolymer is and/or is biodegradable.
  • the polymer scaffold is a hyperbranched structure, such as disclosed in U.S. Patent Application No. 62/737,603, which is incorporated by reference in its entirety for all purposes.
  • the scaffold is a viscosity enhancing agent.
  • the scaffolds, polymers, mixtures of polymers, etc. have structural units (e.g., repeat units, etc.) along a chain of a polymer.
  • the one or more structural units is functionalized with one or more instances of each of R 1 , R 2 , R 3 , R 4 , R 5 , and R6.
  • each instance of R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are independently selected from the group consisting of -OH, -NH 2 , -OCH 3 , -C(O)OH, -CH 2 OH, -CH 2 OCH 3 , -CH 2 OCH 2 CH 2 OH, -OCH 2 C(O)O H, -CH 2 OCH 2 C(O)OH, -CH 2 C(O)OH, -NHC(O)-CH 3 , -C(O)O((CH 2 ) a O) b -H, -C(O)O((CH 2)aO) b -(CH 2 )cH, -C(O)O(C 1 -5alkyl), -C(O)-NH-((CH 2 )dNH)e-H, -C(O)-NH-((CH 2 )dNH)e-( CH 2 ) f H
  • a non-derivatized polymer chain having one or more hydroxyl, amino, or carboxyl functional groups can be functionalized and/or derivatized via those functional groups to add, for example, one or more of R 1 , R 2 , R 3 , R 4 , R 5 , and R6.
  • the disclosed methods are applicable to any biocompatible polymer having one or more of these functional groups pendant from the polymer chain.
  • the polymer is a biopolymer.
  • the polymer is a biodegradable polymer.
  • the polymer is a polysaccharide.
  • the polysaccharide comprises a polymer derived from chitosan, hyaluronic acid, carboxymethylcellulose, hydroxyethyl cellulose, methyl cellulose, cellulose, alginate, cyclodextrin, aminoglycosides, or other polysaccharide.
  • the polysaccharide comprises one or more of the following structures:
  • any one or more of the hydroxyl, amino, or carboxyl functional groups shown above can be functionalized or derivatized via those functional groups to add, for example, one or more of R 1 , R 2 , R 3 , R 4 , R 5 , and R6.
  • any one of the amino groups of an aminoglycoside could be functionalized with a linking unit (as disclosed in PCT/IB2018/052144, published as WO/2018/178902, which is hereby incorporated by reference in its entirety) to prepare a macromolecular structure.
  • the scaffold and/or NO releasing polymer system comprises one or more structural units represented by Formula I:
  • the structural unit represented by Formula I represents one or more of a saccharide unit of a cellulose polymer, a saccharide unit of a hyaluronic acid polymer, a saccharide unit of an alginate polymer, a saccharide unit of a chitosan polymer, a saccharide unit of a carboxymethylcellulose polymer, a saccharide unit of a hydroxyethylcellulose polymer, a saccharide unit of a methyl cellulose polymer, and/or a saccharide unit of a cyclodextrin ring structure.
  • Formula I has the stereochemical configuration shown in Formula I’:
  • the polymer of the scaffold comprises carboxymethylcellulose.
  • the scaffold and/or NO releasing polymer system comprises one or more structural units represented by Formula II:
  • the structural unit represented by Formula I represents one or more of a saccharide unit of a cellulose polymer, a saccharide unit of a hyaluronic acid polymer, and/or a saccharide unit of an alginate polymer.
  • Formula II has the stereochemical configuration shown in Formula II’:
  • the polymer of the scaffold comprises hyaluronic acid.
  • each instance of R 1 , R 2 , R 3 , R 4 , R 5 , and R6 is independently selected from the group consisting of:
  • any one of the secondary amines can be functionalized as an NO donating moiety, including, for example:
  • various structural units e.g., repeat units
  • functionalization of structural units with various moieties
  • levels of crosslinking if crosslinked
  • molecular weight if crosslinked
  • concentrations or other chemical features of the disclosed scaffolds
  • by changing one or more of these features one or more properties of the scaffolds can be tuned.
  • the NO release rate, antimicrobial effect, water solubility, degradation rate, viscosity, gel firmness (where the scaffold forms a gel), viscoelasticity, modulus, etc. are tunable.
  • properties of the polymer and or composition prepared therefrom can be tuned by adjusting the molecular weight of the polymer used.
  • the weight-average molecular weight (Mw) in kDa of polymers disclosed herein are greater than or equal to about: 2.5, 5.0, 7.0, 10, 15, 30, 50, 100, 200, 500, 750, 1,000, 2,000, 10,000, or ranges including and/or spanning the aforementioned values.
  • the number-average molecular weight (Mn) in kDa of polymers disclosed herein are greater than or equal to about: 2.5, 5.0, 7.0, 10, 15, 30, 50, 90, 100, 200, 500, 700, 1,000, 2,000, 10,000, or ranges including and/or spanning the aforementioned values.
  • the polymers disclosed herein may have n repeat units. In several embodiments, n equal to or at least about: 10, 25, 50, 100, 250, 500, 1000, 2500, 5000, 10000, or ranges including and/or spanning the aforementioned values.
  • size exclusion chromatography (SEC) can be used to measure the molecular weight of the scaffold structures disclosed herein.
  • the scaffold structures can be characterized using their polydispersity index.
  • the polydispersity index (PDI) is a measure of the distribution of molecular mass in a given polymer sample. PDI can be calculated by dividing the weight average molecular weight and the number average molecular weight.
  • the scaffold structures have a PDI of greater than or equal to about: 1.05, 1.1, 1.2, 1.3, 1.5, 1.7, 1.8, 1.9, 2.0, or ranges including and/or spanning the aforementioned values.
  • the polymers may be water soluble and/or mutually miscible.
  • the scaffolds are soluble in water (at about 20 °C) at a concentration of greater than or equal to about: 1 mg/ml, 10 mg/ml, 20 mg/ml, 50 mg/ml, 100 mg/ml, 200 mg/ml, 300 mg/ml, 400 mg/ml, 500 mg/ml, or ranges including and/or spanning the aforementioned values.
  • different NO carrying polymers can be combined to prepare aqueous solutions comprising concentrations equal to or at least about: 100 ⁇ g/mL, and can be higher, e.g. about 1 mg/ml, about 5 mg/ml, about 10 mg/ml, about 20/ml, or about 40 mg/ml or higher.
  • the amount of the second polymer in the aqueous composition can be at least about 10% by weight, based on the weight of the first polymer, and may be higher, e.g., at least about 20% by weight, at least about 30% by weight, or at least about 50% by weight, same basis.
  • the polymers in an aqueous composition are selected such the polymers are mutually miscible.
  • the first polymer with antimicrobial activity and the second polymer with antimicrobial activity are considered mutually miscible if at least about 90% of the polymeric components remain mutually soluble 24 hours after mixing and maintaining at room temperature in water at a concentration of each polymer of 1 mg/ml, upon visible examination.
  • Such mutual miscibility of the water polymers can be achieved, despite an expectation of phase separation due to the typical mutual incompatibility of polymers in aqueous solution at the 1 mg/ml concentrations and molecular weights described herein.
  • the aqueous compositions described herein can be prepared by intermixing the individual polymeric components with water, e.g., at room temperature with stirring.
  • the polymers (or mixtures of polymers, etc.) disclosed herein have properties characteristic of a viscous fluid and/or of a gel.
  • the polymers (or mixtures of polymers, etc.) have a gelling point at room temperature (in water or PBS) at a concentration (in w/w %) of less than or equal to about: 0.5%, 1%, 2.5%, 5%, 10%, or ranges including and/or spanning the aforementioned values.
  • the polymers (or mixtures of polymers, etc.) may have a gelling point in water.
  • the polymers gel in water (at about 20 °C) at a concentration of greater than or equal to about: 0.5 mg/ml, 1 mg/ml, 10 mg/ml, 20 mg/ml, 50 mg/ml, 100 mg/ml, 250 mg/ml, or ranges including and/or spanning the aforementioned values.
  • the polymers at a concentration of 5% w/w solution, have a viscosity (in cPa ⁇ s at 20 °C) of equal to or at least about: 10, 50, 100, 1,000, 2,000, 5,000, 10,000, or ranges including and/or spanning the aforementioned values.
  • the polymers have an intrinsic viscosity of equal to or greater than about: 0.5 m 3 /kg, 1.0 m 3 /kg, 2.0 m 3 /kg, 4.0 m 3 /kg, 8.0 m 3 /kg, or ranges including and/or spanning the aforementioned values.
  • the polymers at a concentration of 5% w/w solution, have a firmness of equal to or at least about: 1.0 mN, 2.5 mN, 5 mN, 10 mN, 15 mN, 20 mN, 30 mN, 50 mN, or ranges including and/or spanning the aforementioned values.
  • the polymers at a concentration of 5% w/w solution, have a work of adhesion (in mN*mm) of equal to or at least about: 1.0, 2.5, 5, 10, 15, 20, 30, 50, 100, or ranges including and/or spanning the aforementioned values.
  • the polymers at a concentration of 5% w/w solution, have a storage modulus (G’) in Pa of equal to or at least about: 250, 500, 1,000, 2,000, 4,000, 5,000, 10,000, or ranges including and/or spanning the aforementioned values. In several embodiments, at a concentration of 5% w/w solution, the polymers have an elastic modulus (G”) in Pa of equal to or at least about: 25, 50, 100, 200, 400, 500, 1,000, 2,000, 5,000, 10,000, or ranges including and/or spanning the aforementioned values. In several embodiments, the aqueous composition is characterized by a barrier activity, as measured by a decrease in the diffusion rate of an anionic dye of more than 2 logs at a total scaffold concentration of 40 mg/ml or less.
  • the gels are stable at a variety of temperatures 20 °C (e.g., 40° C, 45° C, 55° C, 60° C, 80° C, etc.) and are stable for prolonged storage periods (e.g., 10 hours, 20 hours, 22 hours, 25 hours, 30 hours, etc., days such as 1 day, 3 days, 5 days, 6 days, 7 days, 15 days, 30 days, 45 days, etc., weeks such as 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, etc., months such as 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, etc., or even years (1 year or greater)).
  • temperatures 20 °C e.g., 40° C, 45° C, 55° C, 60° C, 80° C, etc.
  • prolonged storage periods e.g., 10 hours, 20 hours, 22 hours, 25 hours, 30 hours, etc., days such as 1 day, 3 days, 5 days, 6 days, 7 days, 15 days, 30 days, 45 days, etc
  • the viscosity of the composition increases with increasing temperature, as described above. In several embodiments, the viscosity of the composition decreases with decreasing temperature. For example, if the composition is above the gelling temperature, then the composition has a relatively high viscosity, such as in the form of a gel. In several embodiments, if the composition is cooled to below the gelling temperature, then the composition decreases in viscosity, such as in the form of a liquid.
  • the polymers as disclosed herein may be reversible polymers (e.g., thermoreversible polymers), where the transition from liquid to gel may be reversed upon exposure to appropriate conditions.
  • compositions of the present disclosure include thermoreversible polymers, where the viscosity of the composition may be changed depending on the temperature of the composition.
  • the tunability of the viscosity enables a tailored composition profile upon delivery (e.g., more liquid at a delivery temperature and more viscous at, for example, body temperature).
  • the polymers are characterized by a degree of swelling when exposed to water.
  • the swelling degree % of the polymers disclosed herein is equal to or at least about: 100, 250, 500, 1,000, 2,000, 5,000, or ranges including and/or spanning the aforementioned values.
  • the polymers may swell or otherwise expand by 2X, 4X, 5X, 10X, 20X, 50X, 100X, or more.
  • the polymers disclosed herein have a gelling temperature similar to the normal body temperature of a subject, such as similar to human body temperature, or 37° C.
  • gelling temperature is meant the point on intersection between the plot for the elastic modulus and the plot for the viscous modulus.
  • the composition if the composition is below the gelling temperature, then the composition has a relatively low viscosity, such as in the form of a liquid.
  • the composition if the composition is above the gelling temperature, then the composition increases in viscosity (e.g., polymerizes), such that the composition is in the form of a gel.
  • Compositions that transition from a liquid to a gel may facilitate administration of the composition to the subject, for example by facilitating injection of a low viscosity (e.g., liquid) composition at a temperature below the gelling temperature.
  • the temperature of the composition may increase due to absorption of heat from the surrounding body tissue, such that the composition increases in viscosity (e.g., transitions from a liquid to a gel, or polymerizes), thus providing structural and/or geometric support to the body tissue at the target treatment site.
  • gelling of the composition at the target treatment site may also facilitate retention of the composition at the treatment site by reducing the diffusion and/or migration of the composition away from the treatment site.
  • the composition has a gelling temperature of 30° C to 40° C, such as from 32° C to 40° C, including from 35° C to 40° C. In certain instances, the composition has a gelling temperature of 37° C.
  • the methods disclosed herein provide NO- releasing polymers having NO storage capacities (in ⁇ mol NO/mg polymers) of greater than or equal to about: 0.25, 0.4, 0.5, 1.0, 1.5, 2.0, 3.0, or ranges including and/or spanning the aforementioned values. In some embodiments, within 2 h of being added to a PBS buffer solution as described in the Examples, the NO-releasing polymers, release greater than or equal to about: 25%, 50%, 75%, 85%, 90%, 95%, 100%, or ranges including and/or spanning the aforementioned values, their total wt% of bound NO.
  • NO release in use for reducing or eliminating a biofilm occurs in similar amounts, e.g., about 20-25%, about 30-50%, about 60-75%, at least 80%, at least 85%, at least 90%, at least 95%, ranges including and/or spanning the aforementioned values, of the total wt% of bound NO.
  • the NO release may occur over a period of about 0.01 hours, 0.1 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours, 36 hours, 48 hours, 60 hours, or ranges including and/or spanning the aforementioned values.
  • the NO release half-life is equal to or at least about: 0.01 hours, 0.1 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours, 36 hours, 48 hours, 60 hours, or ranges including and/or spanning the aforementioned values.
  • the NO release occurs in less than or equal to about: 0.01 hours, 0.1 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours, 36 hours, 48 hours, 60 hours, or ranges including and/or spanning the aforementioned values.
  • nitrosamine is not present during NO release.
  • the phrase “nitrosamine is not present” refers to levels of nitrosamine which are not detectable as determined by a UV-vis spectrum (or by other accepted methods in the art).
  • the disclosed scaffolds and/or polymers of the disclosed compositions have a degradation rate per hour in an amylase enzyme exposure assay of less than or equal to about: 0.2%, 0.5%, 1.0%, 1.5%, 2.5%, 5.0%, 10%, or ranges including and/or spanning the aforementioned values.
  • the disclosed functionalized NO-releasing polymers have antimicrobial activity.
  • the disclosed functionalized NO-releasing polymers provide greater than or equal to 90% bacterial reduction in a bacterial viability assay performed under static conditions over 2 hours against one or more of P. aeruginosa, S. aureus P. gingivalis, A. actinomycetemcomitans, A. viscosus, and/or S. mutans at a polymer concentration of equal to or less than about: 8 mg/ml, 6 mg/ml, 4 mg/ml, 2 mg/ml, 1 mg/ml, 0.5 mg/ml, or ranges including and/or spanning the aforementioned values.
  • the disclosed functionalized NO-releasing polymers provide greater than or equal to 99% bacterial reduction and/or a 2 to 3 log reduction in a bacterial viability assay performed under static conditions over 2 hours against a gram positive bacteria at a polymer concentration of equal to or less than about: 8 mg/ml, 6 mg/ml, 4 mg/ml, 2 mg/ml, 1 mg/ml, 0.5 mg/ml, or ranges including and/or spanning the aforementioned values.
  • the disclosed functionalized NO-releasing polymers provide greater than or equal to 99% bacterial reduction and/or a 2 to 3 log reduction in a bacterial viability assay performed under static conditions over 2 hours against a gram negative bacteria at a polymer concentration of equal to or less than about: 8 mg/ml, 6 mg/ml, 4 mg/ml, 2 mg/ml, 1 mg/ml, 0.5 mg/ml, or ranges including and/or spanning the aforementioned values.
  • bacterial reduction is greater than 95%, greater than 98%, or greater than 99%.
  • Cross-links are bonds that link one polymer chain to another (e.g., by covalent bonds or ionic bonds).
  • polymers capable of crosslinking generally exhibit branches off a main chain.
  • a crosslinking agent such as a calcium cation
  • the negatively charged branches from the same or different chains are attracted to the positive cation.
  • the branch joining chains together is referred to as a “crosslink.”
  • crosslinks When polymer chains are linked together by crosslinks, they may lose some of their ability to move as individual polymer chains. For example, a liquid polymer (where the chains are freely flowing) can be turned into a“solid” or“gel” by crosslinking the chains together.
  • an anionic polymer such as sodium alginate is crosslinked with calcium chloride.
  • the sodium alginate is able to be sustained in a solution, but the addition of calcium chloride causes the alginate chains to congregate or crosslink with the calcium cations, thereby forming an immobilized product.
  • Other crosslinkers may also be used, depending on the embodiment.
  • the generally immobilized product may also generally immobilize other materials that may be present such as active agents.
  • crosslinks can be formed by chemical reactions that are initiated by heat, pressure, change in pH, or radiation. For example, mixing of an unpolymerized or partially polymerized resin with specific chemicals called crosslinking reagents can result in a chemical reaction that forms crosslinks.
  • Crosslinking can also be induced in materials that are normally thermoplastic through exposure to a radiation source, such as electron beam exposure, gamma-radiation, or UV light.
  • the polymers disclosed herein can be crosslinked using salts with multiple charges or multifunctional compounds to covalently crosslink the structures (e.g., diamines, triamines, dicarboxylic acids, diepoxides, etc.).
  • Calcium chloride a reagent used in some of the embodiments disclosed herein, provides an example of a simple ionic bond.
  • calcium (Ca) and chlorine (C1) are combined, the calcium atoms each lose two electrons, forming cations (Ca 2+ ), and the chlorine atoms each gain an electron to form anions (C1-). These ions are then attracted to each other in a 1:2 ratio to form calcium chloride (CaCl 2 ).
  • Other cation to anion ratios are also possible depending on the materials used.
  • calcium salts other than calcium chloride could be used as well as other suitable metals such as other multivalent cations.
  • alginates other than sodium alginate may be used such as potassium and ammonium alginates.
  • crosslinking could be used with materials (e.g., polysaccharides) other than alginate.
  • materials e.g., polysaccharides
  • a material that electrostatically cross-links to form a suitable binding material for hemostatic applications can be used.
  • Hydrogels can be synthesized by cross-linking each polymer using an appropriate cross-linking agent chosen according to the chemical moieties present along the polysaccharide chains.
  • amine containing polymers can be crosslinked with carboxylic acid containing polymers by simple coupling reactions (e.g., with EDC, etc.).
  • EDC electrospray
  • hyaluronic acid and carboxymethylcellulose hydrogels can be synthesized following the same chemical route, e.g., by exploiting the EDC chemistry: basically, an amide bond between the carboxylic groups of the polysaccharides and the primary amine of 1,3-diaminopropane (DAP)—the cross-linking agent-can be formed thanks to the presence of EDC.
  • the cross-linking agent, DAP can be added to the mixture at a molar ratio of 0.5 with respect to the moles of carboxylic acid of the polymers and to EDC and NHS moles.
  • the polymers are administered as aqueous gels, e.g., topically.
  • the gels comprise one or more salts and are isotonic.
  • compositions can take the form of, for example, tablets or capsules prepared by a conventional technique with pharmaceutically acceptable excipients, such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycollate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline cellulose or
  • a therapeutic agent can be formulated in combination with hydrochlorothiazide, and as a pH stabilized core having an enteric or delayed release coating which protects the therapeutic agent until it reaches the target organ.
  • the composition includes two or more polymers with a certain ratio (w/w).
  • the ratio (w/w) is 1:10, or 1:9, or 1:8, or 1:7, or 1:6, or 1:5, or 1:4, or 1:3, or 1:2, or 1:1, or 2:1, or 3:1, or 4:1, or 5:1, or 6:1, or 7:1, or 8:1, or 9:1, or 10:1.
  • the ratio (w/w) may range from 1:1 to 10:1, such as 2:1 to 10:1, including 3:1 to 10:1, or 4:1 to 10:1, or 4:1 to 9:1, or 4:1 to 8:1, or 4:1 to 7:1, or 4:1 to 6:1.
  • the ratio (w/w) is 5:1.
  • each polymer of the mixture is provided at a concentration of less than or equal to about: 1 mg/ml, 10 mg/ml, 20 mg/ml, 50 mg/ml, 100 mg/ml, 250 mg/ml, or ranges including and/or spanning the aforementioned values.
  • An unmet need in the area of wound healing, general surgery, and orthopedic surgery is for a antimicrobial material that can form a gel, that can release NO at a requisite rate, and that can degrade during a desired timeframe.
  • This tailored degradation rate can be made to comport with the healing cycle of each specific condition and/or can comport to a time where the wound is at high risk of infection. Examples of these conditions include procedures such as hernia repair, diabetic foot ulcer healing, and orthopedic tendon repairs to name only a few.
  • the compounds and materials disclosed herein are targeted towards compositions that have tailorable degradation times.
  • Some embodiments provide a method for treating a tissue defect comprising positioning any of the polymers described herein at, over, or into the tissue defect.
  • the tissue defect is a wound.
  • Several embodiments provide a method for treating a wound, for performing tissue repair, and/or for providing tissue and organ supplementation.
  • the first step of treating a tissue defect, wound, and/or supplementing and replacing tissue involves identifying a patient in need of an antimicrobial scaffold to aid in the remedying and healing of a tissue defect, healing of a wound, or in need of a tissue supplement.
  • a non-limiting list of patients in need of an antimicrobial scaffold includes patients suffering tissue defects.
  • the patients in need of an antimicrobial scaffold suffer from wounds including those from burns, skin ulcers, lacerations, bullet holes, animal bites, and other wounds prone to infection.
  • Antimicrobial polymers can also be used in the treatment of diabetic foot ulcers, venous leg ulcers, pressure ulcers, amputation sites, in other skin trauma, or in the treatment of other wounds or ailments.
  • Patients in need of an antimicrobial scaffold also include patients in need of repair and supplementation of tendons, ligaments, fascia, and dura mater.
  • Degradable antimicrobial polymers can be used in supplement tissue in procedures including, but not limited to, rotator cuff repair, Achilles tendon repair, leg or arm tendon or ligament repair (e.g., torn ACL), vaginal prolapse repair, bladder slings for urinary incontinence, breast reconstruction following surgery, hernia repair, staple or suture line reinforcement, bariatric surgery repair, pelvic floor reconstruction, dural repair, gum repair, bone grafting, and reconstruction.
  • a patient in need of an antimicrobial scaffold also includes one in need of tissue or organ replacement.
  • the antimicrobial polymers described herein can be used as fillers and/or to supplement and/or replace tissue by acting as an artificial extracellular matrix.
  • an antimicrobial scaffold can be used to support cell and tissue growth. Briefly, cells can be taken from a patient or a viable host and seeded on an antimicrobial scaffold either in vivo or ex vivo. Then as the patient’s natural tissues invade the material, it is tailored to degrade and leave only naturally occurring tissues and cells free of bacterial infection.
  • applications also include delivery of therapeutic molecules to a localized site, use as adhesives or sealants, and as viscosupplements, and in wound healing, among others.
  • the stabilized compositions may also be used as tissue fillers, dermal fillers, bone fillers, bulking agents, e.g., as a urethral or an esophageal bulking agent, and embolic agents as well as agents to repair cartilage defects/injuries and agents to enhance bone repair and/or growth.
  • an antimicrobial scaffold can be placed in or on a patient in, for example, a void space to fill the space.
  • compositions for repairing an injured tissue.
  • the composition is formulated for administration to a target treatment site in a subject.
  • the composition may be formulated to facilitate administration to a damaged or infected tissue in a subject.
  • the composition after administration of the composition (e.g., the antimicrobial scaffold), the composition may increase in temperature due to absorption of heat from surrounding body tissue of the subject.
  • the body temperature of the subject is sufficient to cause the composition to increase in viscosity (e.g., transition from a liquid to a gel.
  • the increase in viscosity e.g., gelling
  • a syringe or catheter may be used to inject the composition in vivo.
  • the composition may be injected directly to the treatment site, or may be allowed to partially pre-heat in the syringe in order to increase the viscosity of the composition prior to injection.
  • a pre-heated formulation may reduce the possibility that a less viscous composition may diffuse and/or migrate away from the tissue area of interest after injection.
  • cariogenic bacteria e.g., Streptococcus mutans, Actinomyces viscosus
  • periodontal pathogens e.g., Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans
  • Oral disease is among the most prevalent health problems faced by humans.
  • Gram-positive cariogenic (e.g., Streptococcus mutans, Actinomyces viscosus) and Gram-negative periodontal (e.g., Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans) bacteria represent the main aggravators associated with the evolution and progression of dental caries and periodontal disease, respectively.
  • Gram-negative periodontal bacteria e.g., Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans
  • Chlorhexidine a common oral antiseptic, can alter taste, stain teeth and tongue, and irritate buccal mucosa.
  • Macromolecule NO-delivering vehicles kill Gram-negative periodontal pathogens.
  • these materials have not been demonstrated to kill Gram-positive cariogenic bacteria at a safe concentration (e.g., a concentration that is bacteriocidal but non-toxic towards mammalian cells).
  • a safe concentration e.g., a concentration that is bacteriocidal but non-toxic towards mammalian cells.
  • the lack of biodegradability and potential cytotoxicity of the silica nanoparticles also hinders their future for biomedical application.
  • Current research also focuses on utilizing nanomaterials including silver, gold, zinc, and copper, as replacement for traditional antibiotics that suffered from fostering bacterial resistance. However, these nanomaterials may accumulate inside the body and may cause accumulative toxicity, limiting their future for certain applications. Developing oral therapeutics that are capable of killing those disease-causing bacteria is important to maintain a healthy oral cavity.
  • the structures disclosed herein e.g., NO scaffolds and/or polymers
  • the compositions disclosed herein may be used as eye drop formulations (e.g., artificial tears).
  • the composition comprises from about 0.1% to about 1.0% of the scaffold (or at a concentration as disclosed elsewhere herein).
  • the mixture comprises more than one type of polymer scaffold (e.g., HA-derived scaffolds and CMC-derived scaffolds) with the second polymer scaffold being present in an amount of 0.05% to about 0.15% (or at a concentration as disclosed elsewhere herein).
  • Cystic fibrosis is a genetic disorder characterized by poor mucociliary clearance and chronic bacterial infections.
  • NO nitric oxide
  • Treatment with NO limits bacterial resistance due to its multiple biocidal mechanisms (e.g., induction of nitrosative and oxidative stress). It has surprisingly been found that by storing NO on a scaffold using one of the disclosed designs, bactericidal efficacy is improved and systemic cytotoxicity is reduced. Treatments are effective against planktonic and biofilm-based pathogens, and cytotoxicity assays against mammalian lung cells demonstrate little harm to a treated subject’s cells.
  • CF is a debilitating disease characterized by chronic bacterial infection of the lungs, resulting in life expectancies as low as two decades.
  • a genetic defect in the CF transmembrane conductance regulator (CFTR) impedes the normal transport of ions (e.g., Cl-) to the airway surface liquid, inhibiting water transport.
  • ions e.g., Cl-
  • the airway epithelium dehydrates, creating thickened mucus that can no longer be efficiently cleared via mucociliary clearance mechanisms.
  • goblet cells continually excrete mucins into the dehydrated airway, mucus accumulation is accelerated to the point where the cilia become damaged, or nonfunctional, and are unable to clear mucus from the airway.
  • Planktonic bacteria thrive in this static environment, promoting the formation of complex communities of pathogenic bacteria known as biofilms.
  • the exopolysaccharide matrix produced by these biofilms inhibits oxygen diffusion, creating pockets of anaerobic environments and altering bacterial metabolism. This combination of a concentrated mucus layer and robust biofilms severely decreases the antibacterial efficacy of common CF therapies.
  • the microbial load to be reduced and/or eliminated comprises drug-resistant bacteria.
  • the drug-resistant bacteria comprise carbapenem-resistant Enterobacteriaceae.
  • the drug-resistant bacteria comprise Methicillin-resistant Staphylococcus aureus.
  • the microbe comprises human immunodeficiency virus, herpes simplex virus, papilloma virus, parainfluenza virus, influenza, hepatitis, Coxsackie Virus, herpes zoster, measles, mumps, rubella, rabies, pneumonia, (hemorrhagic viral fevers, H1N1, and the like), prions, parasites, fungi, mold, yeast and bacteria (both gram-positive and gram-negative) including, among others, Candida albicans, Aspergillus niger, Escherichia coli (E. coli), Pseudomonas aeruginosa (P.
  • microorganism and microbe can include wild-type, genetically-engineered or modified organisms.
  • the formulations and methods disclosed herein are for topical use or treatment of a surface, such as the oral mucosa.
  • the scaffolds and/or compositions thereof may be administered by direct injection or application to, for example, an injured tissue. Suitable routes also include injection or application to a site adjacent to the injured tissue. Administration may include parenteral administration (e.g., intravenous, intramuscular, or intraperitoneal injection), subcutaneous administration, administration into vascular spaces, and/or administration into joints (e.g., intra-articular injection). Additional routes of administration include intranasal, topical, vaginal, rectal, intrathecal, intraarterial, and intraocular routes.
  • the scaffolds and compositions disclosed herein can be applied as a gel to a site of treatment. In several embodiments, the scaffolds and compositions can be applied as a liquid.
  • liquid preparations for oral or topical administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or another suitable vehicle before use.
  • Such liquid preparations can be prepared by conventional techniques with pharmaceutically acceptable additives, such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats
  • emulsifying agents e.g., lecithin or acacia
  • non-aqueous vehicles e.g., almond oil, oily esters, e
  • compositions also can contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
  • Preparations for oral administration can be suitably formulated to give controlled release of the active compound.
  • buccal administration the compositions can take the form of tablets or lozenges formulated in a conventional manner.
  • the disclosed compounds also can be formulated as a preparation for implantation or injection.
  • the compounds can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).
  • suitable polymeric or hydrophobic materials e.g., as an emulsion in an acceptable oil
  • ion exchange resins e.g., as sparingly soluble derivatives (e.g., as a sparingly soluble salt).
  • the compounds also can be formulated in rectal compositions (e.g., suppositories or retention enemas containing conventional suppository bases, such as cocoa butter or other glycerides), creams or lotions, or transdermal patches.
  • compositions also are provided which are suitable for administration as an aerosol by inhalation.
  • the polymer structures described herein are formulated in solution and/or aerosol form.
  • these formulations comprise a solution or suspension of a polymers described herein.
  • the desired formulation can be placed in a small chamber and nebulized. Nebulization can be accomplished by compressed air or by ultrasonic energy to form a plurality of liquid droplets or solid particles comprising the NO-releasing hyper- branched polyamidomines.
  • the presently disclosed NO-releasing hyper- branched polyamidomines can be administered via inhalation to treat bacterial infections related to cystic fibrosis.
  • Cystic fibrosis-related bacterial infections include, but are not limited to stenotrophomonis, mybacterium avium intracellulaire and m. abcessus, burkhoderia cepacia and Pseudomonas aeruginosa (P. aeruginosa) infections.
  • the subject matter described herein is directed to the following embodiments: 1.
  • R 1 , R 2 , and R 3 are independently selected from the group consisting of -OH, -CH 2 OH, -OCH 2 C(O)OH, -CH 2 OCH 2 C(O)OH, -C(O)-O-((CH 2 )aO) b -H, -C (O)-O-((CH 2 ) a O) b -(CH 2 ) c H, -C(O)-O-(C 1-5 alkyl), -C(O)-NH-((CH 2 ) d NH) e -H, -C(O )-NH-((CH 2 ) d NH) e -(CH 2 ) f H, -CH 2 C(O)-NH-((CH 2 ) d NH) e -H, -CH 2 C(O)-NH-((CH 2 )dNH)e-(CH 2 ) f H, -C(O)-
  • each instance of X 1 , X 2 , and X 3 is independently selected from -O-, -S-, -NH- , C(O)NH-;
  • At least one of X 1 , X 2 , and X 3 is represented by one of the following:
  • the compound has a viscosity of equal to or at least about 10 mPa ⁇ s at 20 °C at a concentration of 5% w/w in water.
  • R 1 is -CH 2 C(O)-X 1 -((CH 2 ) g X 2 ) h ((CH 2 ) j X 3 )k-(CH 2 ) 1 H.
  • An NO releasing hyaluronic acid-derived polymer compound comprising a unit structure of Formula II:
  • R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are independently selected from the group consisting of of -OH, -NH 2 , -CH 2 OH, -C(O)OH, -NHC(O)-CH 3 , -O-((CH 2 )aO) b -H, -O-((CH 2 )aO ) b -(CH 2 )cH, -O-(C 1 -5alkyl), -NH-((CH 2 )dNH)e-H, -NH-((CH 2 )dNH)e-(CH 2 ) f H, -X 1 -( (CH 2 ) g X 2 ) h -H, -X 1 -((CH 2 ) g X 2 ) h -(CH 2 ) 1 H, -CH 2 C(O)-X 1 -((CH 2 ) g X 2 ) h (
  • each instance of a, b, c, d, e, f, g, h, i, j, k, and l is independently selected from an integer of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
  • each instance of X 1 , X 2 , and X 3 is independently selected from -O-, -S-, -NH- , C(O)NH-;
  • At least one of X 1 , X 2 , and X 3 is represented by one of the following:
  • the compound has a viscosity of equal to or at least about 10 mPa ⁇ s at 20 °C at a concentration of 5% w/w in water.
  • R 1 is -CH 2 C(O)-X 1 -((CH 2 ) g X 2 ) h ((CH 2 ) j X 3 )k-(CH 2 ) 1 H.
  • a viscosity enhancing agent comprising:
  • a scaffold comprising a polymer having structural units along a chain of the polymer, one or more structural units being functionalized with one or more instances of each of R 1 , R 2 , and R 3 ;
  • R 1 , R 2 , and R 3 are independently selected from the group consisting of -OH, -NH 2 , -OCH 3 , -C(O)OH, -CH 2 OH, -CH 2 OCH 3 , -CH 2 OCH 2 CH 2 OH, -OCH 2C(O)OH, -CH 2 OCH 2 C(O)OH, -CH 2 C(O)OH, -NHC(O)-CH 3 , -C(O)O((CH 2 ) a O) b - H, -C(O)O((CH 2 )aO) b -(CH 2 ) c H, -C(O)O(C 1 -5alkyl), -C(O)-NH-((CH 2 )dNH)e-H, -C( O)-NH-((CH 2 ) d NH) e -(CH 2 ) f H, -CH 2 C(O)-NH-(
  • each instance of a, b, c, d, e, f, g, h, i, j, k, and l is independently selected from an integer of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
  • each instance of X 1 , X 2 , and X 3 is independently selected from -O-, -S-, -NH- , C(O)NH-;
  • At least one instance of X 1 , X 2 , and X 3 is represented by one of the following:
  • the scaffold has a viscosity of equal to or at least about 10 mPa ⁇ s at 20 °C at a concentration of 5% w/w.
  • G 1 is selected from the group consisting of:
  • G 2 is selected from the group consisting of:
  • each instance of R 1 is independently selected from the group consisting of -H, optionally substituted C 1 -C 6 alkyl, optionally substituted polyamino having 1 to 6 repeat units with intervening C 1 -C 6 alkyl groups, optionally substituted polyether having 1 to 6 repeat units with intervening C 1 -C 6 alkyl groups, or indicates a covalent bond to a linking unit;
  • X a is independently selected from -H, -OH, and C 1 - C 6 alkyl
  • R 1 indicates a covalent bond to one or more linking units selected from the following:
  • each instance of W 1 is independently selected from one or more additional aminoglycosides or one or more end-capping substituents and at least one linking unit provides a covalent bridge from the first aminoglycoside to a second aminoglycoside;
  • each instance of R a is independently selected from the group consisting of optionally substituted C 1 -C 6 alkyl, optionally substituted polyamino having 1 to 6 repeat units (with C 1 -C 6 alkyl(s)), or optionally substituted polyether having 1 to 6 repeat units (with C 1 -C 6 alkyl(s)); and
  • the one or more end-capping substituents where present, independently have a formula of -X 1 -((CH 2 ) h X 2 ) i -(CH 2 ) j H.
  • a method of delivering nitric oxide to a subject in need of treatment comprising: administering an effective amount of the compounds or viscosity inducing agents of any one of embodiments 1 to 45 to the subject.
  • a method of treating a disease state comprising:
  • a disease state is selected from the group consisting of a cancer, a cardiovascular disease, a microbial infection, platelet aggregation and platelet adhesion caused by the exposure of blood to a medical device, pathological conditions resulting from abnormal cell proliferation, transplantation rejections, autoimmune diseases, inflammation, vascular diseases, scar tissue, wound contraction, restenosis, pain, fever, gastrointestinal disorders, respiratory disorders, sexual dysfunctions, and sexually transmitted diseases.
  • a pharmaceutical formulation comprising:
  • a method of reducing or preventing microbial load on a surface comprising, applying the compounds or viscosity inducing agents of any one of embodiments 1 to 45 to a surface contaminated with a plurality of microbes; wherein the compounds or viscosity inducing agents of any one of embodiments 1 to 45 generate nitric oxide and induce oxidative and/or nitrosative damage to microbial DNA and membrane structures, thereby preventing or reducing microbial load, and wherein said plurality of microbes comprises two or more of the following: gram-positive bacteria, gram-negative bacteria, fungi, yeast, and viruses.
  • the microbial load comprises microbes associated with the presence of one or more of human immunodeficiency virus, herpes simplex virus, papilloma virus, parainfluenza virus, influenza, hepatitis, Coxsackie Virus, herpes zoster, measles, mumps, rubella, rabies, pneumonia, hemorrhagic viral fevers, H1N1, prions, parasites, fungi, mold, Candida albicans, Aspergillus niger, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, Group A streptococci, S.
  • the following examples pertain to the synthesis of N-diazeniumdiolate functionalized nitric oxide (NO)-releasing hyaluronic acid with tunable NO storage and release kinetics.
  • This embodiment has the following features, advantages, and/or uses.
  • the water solubility and biocompatibility of these scaffolds are high at high molecular weight ( ⁇ 90 kDa) and low molecular weight ( ⁇ 6 kDa) of the NO-release scaffold.
  • theses scaffolds may be useful in the treatment for chronic wounds with the NO-releasing hyaluronic acid derivatives used for antibacterial therapy and cell proliferation.
  • these scaffolds could also be used as a therapeutic for cystic fibrosis with the NO-releasing material acting as an antibacterial agent.
  • TLB Tryptic soy broth
  • TSA tryptic soy agar
  • Pseudomonas aeruginosa P. aeruginosa ; ATCC #47085
  • Escherichia coli E . coir
  • ATCC #43888 Staphylococcus aureus ( S . aureus ; ATCC #29213), Enterococcus faecalis ⁇ E. faecalis, ATCC #29212), multi drug-resistant 1 ⁇ aeruginosa (ATCC #BAA-2110), and methicillin-resistant S. aureus (MRSA; ATCC #33591) were obtained from the American Type Tissue Culture Collection (Manassas, VA).
  • Argon (Ar) carbon dioxide (CO 2 ), nitrogen (N2), oxygen (O 2 ), nitric oxide (NO) calibration (25.87 ppm balance N2), and pure NO (99.5%) gas cylinders were purchased from Airgas National Welders (Raleigh, NC). Distilled water was purified to a resistivity of 18.2 MW cm and a total organic content of ⁇ 6 ppb using a Millipore Milli-Q UV Gradient A10 system (Bedford, MA).
  • Hyaluronic acid (90 kDa or 6 kDa) materials were modified with either N- propyl-l,3-propanediamine (PAPA), A-(2-hydroxyethyl)ethylenediamine (HEDA), bis(3- aminopropyl)amine (DPTA), or diethylenetriamine (DETA) (Scheme la). Briefly, HA (1 g) was dissolved in 40 mL (6 kDa HA) or 100 mL (90 kDa HA) of distilled water.
  • PAPA N- propyl-l,3-propanediamine
  • HEDA A-(2-hydroxyethyl)ethylenediamine
  • DPTA bis(3- aminopropyl)amine
  • DETA diethylenetriamine
  • a 4 1 molar ratio of 1 -ethyl-3 -(3 -dimethylaminopropyl)carbodiimide hydrochloride (EDC) and A-hydroxysuccinimide (NHS), with respect to the carboxylic acid moieties on the HA scaffold, was added, and the solution was titrated to a pH of 3.0 using 0.5 M HC1.
  • GPC Gel permeation chromatography
  • HA90 and HA6 1 H NMR (600 MHz, D 2 O, d) 2.00 (NHC(O)CH 3 ), 3.50 (CHCH 2 OH), 3.60-4.50 (OCHCH(OH)CH(OH)), (OCHCH(OH)CH(OH), 4.50-4.60 (NHCOCH), 5.30 (OCH(CHOH)O), 6.30 (NHCHCH(OH)). 13 C NMR (600 MHz, D 2 O, d) 24.0 (NHC(O)CH 3 ), 65.0 (CHCH 2 OH), 70.0-81.0
  • HA90-DETA and HA6-DETA 1 H NMR (600 MHz, D 2 O, d) 2.60-3.30 (CH 2 CH 2 NHCH 2 CH 2 NH 2 ), 2.00 (NHC(O)CH 3 ), 3.50 (CHCH 2 OH), 3.60-4.50
  • HA90-DPTA and HA6-DPTA 1 H NMR (600 MHz, D 2 O, d) 1.70-1.80 (CH 2 CH 2 CH 2 NHCH 2 CH 2 CH 2 NH 2 ), 2.00 (NHC(O)CH 3 ), 2.50-2.40
  • HA90-PAPA and HA6-PAPA 1 H NMR (600 MHz, D2B& f$ *(2*'*(3* (NHCH 2 CH 2 CH 3 ), 1.40-1.50 (NHCH 2 CH 2 CH 3 ), 1.70-1.80
  • HA90-HEDA and HA6-HEDA 1 H NMR (600 MHz, D 2 O, d) +(1/ (C(O)NHCH 2 CH 2 CH 2 NH), 2.50-3.20 (C(O)NHCH 2 CH 2 CH 2 NH), 2.70-3.50
  • Enzymatic degradation of alkylamine-modified and NO-releasing HA was carried out using a procedure adapted from Turner et al. Briefly, 50 mg of HA90, amine-modified HA90, or NO-releasing HA90 was dissolved in 5 mL of pH 5.0 buffer containing 0.15 M NaCl, 0.1 M CH 3 COONa, and 1 mM Na2EDTA at 37 °C for 30 min with magnetic stirring. Hyaluronidase (2.5 mg) was dissolved in 1 mL of the same buffer and added directly to the HA solution. The mixture was incubated at 37 °C with vigorous stirring for 30 min.
  • this scaffold may be useful in the treatment of bacterial infections.
  • Periodontal diseases encompass a class of inflammatory infections of the gums and surrounding tissue affecting a significant portion of the population. Disease progression is caused by a shift in the microbial composition of healthy dental plaque biofilms resulting in an overabundance of Gram-negative bacteria.
  • the Gram-negative bacteria such as Porphyromonas gingivalis, induce inflammation in dental tissue (e.g., gums, periodontal ligaments, and alveolar bone tissue) leading to the development of periodontal pockets in which these bacteria can continue to thrive. If left untreated, chronic periodontitis eventually results in oral tissue degradation including tooth and bone resorption.
  • studies have shown connections between periodontitis and other systemic inflammatory conditions including cardiovascular disease, coronary heart disease, and adverse pregnancy outcomes, attributable to the spread of pathogenic gram-negative oral bacteria to the bloodstream and other areas of the body.
  • SRP scaling and root planing
  • GCF gingival crevicular fluid
  • Nitric oxide is an endogenous antibacterial agent that plays a key role in the mammalian immune response. It demonstrates broad spectrum antibacterial activity due to its multitude of killing mechanisms involving the formation of reactive byproducts capable of exerting both nitrosative and oxidative stress on bacteria. As a result, NO is less likely to foster bacterial resistance than many conventional antibacterial agents. Challenges in effective localized delivery of gaseous NO have necessitated the development of NO donors capable of storing and releasing nitric oxide. Among the numerous NO donors that have been developed, N-diazeniumdiolates represent an attractive option for dental therapeutics due to their proton-mediated release of NO in aqueous environments.
  • Carboxymethylcellulose (CMC), a water-soluble synthetic derivative of cellulose, is an attractive scaffold for use in the periodontal pocket due to its biocompatibility, adhesivity, and high solution viscosity. It has been widely used as a thickening and stabilizing additive in many industries and has also seen utility in dental applications. Thus, CMC can enhance retention in the periodontal pocket while its water- solubility enables natural clearance over time. Further, CMC can be produced with a range of molecular weights and degrees of substitution, enabling additional tunability toward its intended end use. The presence of carboxylic acid moieties allows for effective modification of the polymer in aqueous solution under mild reaction conditions, which is critical to impart antibacterial properties to the scaffold.
  • N-diazeniumdiolate NO donors on the CMC polymer backbone it must be chemically modified with secondary amine moieties.
  • Carbodiimide crosslinking reactions using EDC and NHS are effective for aqueous modifications of carboxylic acid-containing polysaccharides as demonstrated previously, enabling modification of CMC with four alkylamines (Scheme 2A).
  • N-diazeniumdiolates can be formed by exposing the polymer to high pressures of gaseous NO under basic conditions (Scheme 2B).
  • CMC Carboxymethylcellulose
  • DETA diethylenetriamine
  • DPTA bis(3-aminopropyl)amine
  • HEDA N-(2- hydroxyethyl)ethylenediamine
  • PAPA N-propyl-1,3-propanediamine
  • EDC 1-Ethyl-3- (3-dimethylaminopropyl)carbodiimide
  • NHS N-hydroxysuccinimide
  • Porphyromonas gingivalis strain A7436 was provided by the UNC School of Dentistry, Chapel Hill, NC. Brain heart infusion (BHI) broth and agar, CDC anaerobe 5 vol % sheep blood agar, and GasPak EZ campy sachets were purchased from Becton, Dickinson, and Company (Franklin Lakes, NJ). Wilkins-Chalgren (W _ C) broth was purchased from Thermo Fisher Scientific (Waltham, MA). Human gingival fibroblasts (HGF-1) and FibroLife S2 media were purchased from Lifeline Cell Technology LLC (Frederick, MD).
  • nitric oxide 99.5%
  • argon nitrogen
  • anaerobic gas mixture 10% hydrogen, 5% carbon dioxide, balance nitrogen
  • MTS reagent was purchased from BioVision (Milpitas, CA) and phenazine methosulfate (PMS) was purchased from Millipore Sigma.
  • Common laboratory salts and solvents were purchased from Thermo Fisher Scientific (Waltham, MA).
  • % solutions of CMC were prepared in the mobile phase buffer consisting of 0.1 M acetate (pH 4.6) with 0.1 M NaN03 and 0.02 wt% NaN3. A 50 mL aliquot of each sample was injected and run through two columns in series (2x Shodex OHpak LB804, Showa Denko America, New York, NY) at a flow rate of 0.6 mL min-1.
  • Nitric oxide release from CMC scaffolds was characterized using a Zysense 280i Nitric Oxide Analyzer (NOA, Zysense, Frederick, CO).
  • NOA Zysense 280i Nitric Oxide Analyzer
  • 1 mg NO-releasing sample was submerged in 25 mL deoxygenated PBS (10 mM, pH 7.4, 37 oC). Nitrogen gas was flowed through the solution at 200 mL min-1 to transport NO released from the scaffold to the NOA. Release was measured until NO levels fell below 10 ppb NO per mg scaffold.
  • CMC-DETA diethylenetriamine
  • DPTA bis(3-aminopropyl)amine
  • HEDA N- (2-hydroxyethyl)ethylenediamine
  • PAPA N-propyl-1,3-propanediamine
  • Nitric oxide-release capabilities were imparted onto CMC-amines through the formation of N- diazeniumdiolates. These modifications were achieved by reacting the scaffolds with pressurized (10 bar) NO gas in basic aqueous solution for 3 d. Successful N- diazeniumdiolate formation was confirmed using UV-vis spectroscopy in order to verify the presence of a characteristic absorbance peak at 250 nm ( Figure 21). Further, NO release characteristics were determined in real-time using a chemiluminescence-based nitric oxide analyzer. The full extent of NO release characterization under physiological conditions (37 oC, pH 7.4), shown in Table 6, demonstrates the range of kinetics attainable for CMC- amines as a function of the chemical structure of the amine modification.
  • CMC-DETA/NO possesses the longest half-life ( ⁇ 3 h) as a result of primary amine stabilization of diazeniumdiolate stemming from intramolecular ring formation.
  • CMC-DPTA/NO has similar primary amine interaction with the diazeniumdiolate, but its longer chain length results in a less favorable intramolecular stabilization and thus has a shorter NO-release half-life.
  • CMC-HEDA/NO has a similar half-life to that of CMC-DPTA/NO despite the lack of a primary amine.
  • Example 1 The scaffolds generated in Example 1 are used in the following experiments against various bacterial cultures:
  • control (non-NO-releasing) HA, NO-releasing HA, or neomycin sulfate were dissolved in PBS and titrated with 1 M HCl to adjust the pH to 7.4. Samples were added to a 96-well polystyrene plate and serially diluted in PBS so that each well contained 100 ⁇ L of control HA, NO-releasing HA, or neomycin.
  • Bacterial solution containing 10 6 CFU mL -1 (100 ⁇ L; 1 vol% TSB supplemented PBS) was added to each well, giving final HA concentrations in the range of 0.25 to 32 mg mL -1 or neomycin concentrations from 0.5 to 1024 mg mL -1 .
  • the 96-well plate was then incubated at 37 °C for 4 h with gentle shaking. Untreated bacterial solutions were included in each experiment to ensure bacteria viability over the 4 h duration.
  • bacterial solutions were serially diluted (10-, 100-, and 1000-fold dilutions), spiral plated on TSA plates using an Eddy Jet spiral plater (IUL; Farmingdale, NY), and incubated overnight at 37 °C.
  • Viability of bacteria following treatment with HA or neomycin was determined using a Flash & Go colony counter (IUL; Farmingdale, NY).
  • the minimum bactericidal concentration after a 4 h exposure period (MBC 4h ) was defined as the minimum concentration required to achieve a 3 -log reduction (>99.9% reduction) in bacterial viability relative to untreated bacteria (i.e., reduced bacterial counts from 10 6 to 10 3 CFU mL 1 ).
  • the limit of detection for this counting method is 2.5 x 10 3 CFU mL -1 .
  • the NO dose required for bactericidal action was calculated by multiplying the MBC 4h of the NO-releasing HA samples (mg mL 1 ) with the total NO released in PBS (pH 7.4; mmol NO mg 1 HA) at 4 h.
  • the minimum bactericidal concentrations (MBC 4h ) of NO-releasing hyaluronic acid against various bacteria are set forth in Tables 7 and 9.
  • DPTA-modified HA eradicates all bacteria strains at a dose of ⁇ 2 mg mL 1 .
  • the doses of NO or neomycin required to elicit a 3-log reduction in bacteria viability following 4 h treatment is set forth in Tables 8 and 10.
  • the antibacterial efficacy of active ingredients NO-releasing hyaluronic acids against the various bacteria are set forth in Figures 10 and 13-15. NO is bactericidal against antibiotic- resistant bacteria at low concentrations. All data presented are from n > 3 separate experiments.
  • MBC 4h Minimum bactericidal concentrations (MBC 4h ) of NO-releasing hyaluronic acid against Gram-negative ( E . coli and P. aeruginosa ) and Gram-positive (S. aureus and E. faecalis) bacteria/' _
  • HA-DPTA/NO eradicates bacteria quickly and at low concentrations.
  • Biofilm eradication assay Bacterial cultures of P. aeruginosa and MDR-P. aeruginosa were grown from frozen (-80 °C) stocks overnight in TSB (3 mL) at 37 °C and recultured in fresh TSB to a concentration of 10 8 CFU mL -1 . An aliquot of the 10 8 solution (18 ⁇ L) was added to 1800 ⁇ L of fresh TSB in a 24-well polystyrene plate and incubated at 37 °C with gentle shaking for 72 h.
  • Nitric oxide-releasing DPTA-modified HA or neomycin was dissolved in PBS (750 ⁇ L, pH 7.4, 10 mM) in 1-dram vials and adjusted to pH 7.4 with 1 M HCl. Biofilms (250 ⁇ L) were rinsed with PBS (pH 7.4, 10 mM) and added to the 1-dram vials. Treatment with 4-32 mg mL -1 of NO-releasing DPTA-modified HA or 30-240 ⁇ g mL -1 neomycin sulfate occurred for 24 h at 37 °C with gentle shaking. Untreated biofilms were included in each experiment to ensure biofilm viability over the 24 h duration.
  • biofilms 100 ⁇ L were diluted 10-fold and dispersed via pipetting and vortexing. Biofilm solutions were further diluted (1,000- and 100,000-fold), plated on TSA plates using an Eddy Jet spiral plater, and incubated overnight at 37 °C. Biofilm viability following treatment with HA or neomycin was determined using a Flash & Go colony counter.
  • the minimum biofilm eradication concentration after a 24 h exposure period (MBEC 24h ) was defined as the minimum concentration required to achieve a 5-log reduction (>99.999% reduction) in bacterial viability relative to untreated bacteria (i.e., reduced bacterial counts from 10 8 to 10 3 CFU mL -1 ).
  • the NO dose required for biofdm eradication was calculated by multiplying the MBEC 24h of the NO-releasing HA samples (mg mL -1 ) with the total NO released in pH 7.4 PBS (mmol NO mg -1 HA).
  • the biofdm viability results after 24h treatment of bacteria pre-existing biofdms with neomycin sulfate or Nitric oxide-releasing DPTA-modified HA is set forth in Figures 11, 16, and 17. All data presented are from n > 3 separate experiments. Low molecular weight HA > high molecular weight HA NO > neomycin.
  • L929 murine fibroblasts were grown in DMEM supplemented with 10 vol% FBS and 1 wt% penicillin streptomycin. Cells were incubated in 5 vol% CO 2 under humidified conditions at 37 °C. After reaching 80% confluency, cells were seeded onto 96-well polystyrene plates at a density of 1 c 10 4 cells well -1 . After 24 h incubation at 37 °C, the supernatant was then aspirated and replaced with 100 mL of either control or NO-releasing HA in fresh grown medium with HA concentrations ranging from 0.25 to 32 mg mL -1 .
  • Example 2 The scaffolds generated in Example 2 are used in the following experiments against various bacterial cultures: [00213] Determination of 2 h minimum bactericidal concentrations (MBC2h). P. gingivalis was reinoculated overnight from a frozen stock in W-C anaerobic broth in an anaerobic chamber (Coy Laboratory Products, Grass Lake, MI). A 300 ⁇ L aliquot of bacteria was transferred into fresh broth and grown to 10 8 cfu/mL. A. actinomycetemcomitans was prepared similarly using brain heart infusion (BHI) broth under microaerophilic conditions using a GasPak EZ campy container system.
  • BHI brain heart infusion
  • Bacterial concentrations were confirmed by measuring optical density at 600 nm (OD 600 ). Bacteria were diluted to 10 6 cfu/mL in 1% broth-supplemented PBS 7.4 and exposed to control (i.e., non-NO-releasing) and NO-releasing materials under aerobic conditions for 2 h at 37 °C. Bacteriostatic conditions were confirmed for both bacteria when exposed to no material. After the exposure, samples were diluted 10-1000x in PBS 7.4 and plated on their corresponding agar using an IUL Instruments Eddy Jet 2 spiral plater (Neutec Group, Farmingdale, NY). P. gingivalis on CDC anaerobe 5 vol% sheep blood agar were incubated in the anaerobic chamber for 3 d and A.
  • actinomycetemcomitans on brain heart infusion agar were incubated for 3 d under microaerophilic conditions. After incubation, bacterial concentrations were determined using the plate counting method with an IUL Instruments Flash & Go (Neutec Group, Farmingdale, NY).
  • the NO-releasing scaffolds possessed similar killing efficacy against both pathogens.
  • the control (i.e., non- NO-releasing) scaffolds demonstrated minimal bactericidal action, supporting that NO is indeed acting as the bactericidal agent.
  • the slower NO-release half-life of CMC-DETA/NO resulted in lower NO totals at 2 h, the length of the exposure, thus requiring higher scaffold concentrations to elicit bactericidal activity.
  • the A7436 strain of P. gingivalis possesses a capsule which may reduce NO penetration relative to A. actinomycetemcomitans, resulting in slightly higher MBC2h for three of the tested polymers. Table 11. Antibacterial efficacy of NO-releasing CMC against prominent periodontopathogens
  • HGF-1 cells were cultured in FibroLife S2 media supplemented with 1% penicillin and streptomycin and incubated at 37 °C in humidified 5 vol% CO 2 . Upon reaching ⁇ 80% confluency, cells were trypsinized and placed into tissue-culture treated 96-well polystyrene plates at a density of 10 4 cells/well. After 24 h of incubation in 96-well plates, media was aspirated and replaced with 100 ⁇ L media containing control and NO-releasing CMC.
  • Toxicities of the CMC scaffolds against mammalian cells were evaluated by exposing human gingival fibroblasts (HGF) to both NO-releasing and non-NO-releasing materials for 24 h in Figure 20. Metabolic activity was determined at the endpoint using an MTS assay and correlated to cell viability, with untreated cells representing 100% viability. For the amine-modified CMCs, only CMC-DPTA resulted in significant decreases in HGF viability, decreasing to ⁇ 80% at 8 mg/mL and ⁇ 30% at 16 mg/mL. More significant differences were observed for the NO-releasing CMC polymers.
  • CMC- DETA/NO resulted in consistently higher apparent cell viabilities than baseline, either as a result of proliferative effects of NO or increased cell metabolism.
  • cell viability decreased for all materials, likely in response to the increasing NO doses exerting oxidative and nitrosative stress on the fibroblasts.
  • high cell viability is maintained for 24 h at the MBC2h of all scaffolds with the exception of CMC-DETA/NO.
  • a solution of CMC-DETA as disclosed in Example 2 is prepared at a concentration of 10 mg/ml in PBS.
  • the solution is injected via a syringe into an open laceration from a knife wound of an adult male patient. Upon reaching the internal surfaces of the wound, the viscous solution forms a firm gel. The gel is covered with a bandage.
  • a control another patient with a similar wound is treated by flushing the wound, application of bacitracin application and covering it with a bandage. The experimental and control antibacterial formulations are reapplied and the new bandages are applied daily.
  • the experimental patient shows no sign of infection.
  • the control patient has redness around the wound, indicative of bacterial invasion.
  • a solution of HA6-DPTA/NO as disclosed in Example 1 is prepared at a concentration of 5 mg/ml in PBS.
  • a solution of CMC-DETA as disclosed in Example 2 is prepared at a concentration of 10 mg/ml in PBS.
  • the two solutions are mixed and form a firm gel. This gel is applied to a diabetic foot sore that is infected with MRSA and that has previously been treated with antibiotics. The gel is covered with a bandage. The gel is reapplied along with a fresh bandage daily.
  • actions such as“administering an NO-donating composition” include “instructing the administration of an NO-donating composition.”
  • actions such as“administering an NO-donating composition” include “instructing the administration of an NO-donating composition.”
  • ranges disclosed herein also encompass any and all overlap, sub- ranges, and combinations thereof.
  • Language such as“up to,”“at least,”“greater than,”“less than,”“between,” and the like includes the number recited. Numbers preceded by a term such as“about” or“approximately” include the recited numbers. For example,“about 10 one millipascal-second” includes“10 one millipascal-second.”

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